WO2016049251A1 - Administration, utilisation et applications thérapeutiques de systèmes crispr-cas et compositions de modélisation de mutations dans des leucocytes - Google Patents

Administration, utilisation et applications thérapeutiques de systèmes crispr-cas et compositions de modélisation de mutations dans des leucocytes Download PDF

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Publication number
WO2016049251A1
WO2016049251A1 PCT/US2015/051815 US2015051815W WO2016049251A1 WO 2016049251 A1 WO2016049251 A1 WO 2016049251A1 US 2015051815 W US2015051815 W US 2015051815W WO 2016049251 A1 WO2016049251 A1 WO 2016049251A1
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leukocyte
cas9
express
leukocytes
vector
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PCT/US2015/051815
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English (en)
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Aviv Regev
Oren PARNAS
Marko JOVANOVIC
Nir Hacohen
Thomas EISENHAURE
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The Broad Institute Inc.
Massachusetts Institute Of Technology
The General Hospital Corporation
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Publication of WO2016049251A1 publication Critical patent/WO2016049251A1/fr
Priority to US15/468,652 priority Critical patent/US11197467B2/en

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Definitions

  • the present invention generally relates to the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas System and components thereof. More specifically, the present invention relates to modulating leukocyte activity, identifying genes or genomic elements associated with leukocyte responses, and modeling aberrant leukocyte responses and diseases associated with leukocytes by introducing mutations into the genome of leukocytes ex vivo and/or in vivo using a transgenic CRISPR-Cas9 animal (e.g., mouse) model, including the model(s) and methods for generation and uses thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • RNAi which until recently was the main available tool in mammals, is limited by off-target effects and lack of sufficient suppression of gene expression (Echeverri et al., 2006), whereas more effective strategies based on haploid cell lines (Carette et al., 2009) are not applicable to the diversity of primary cell types and their specialized circuitry.
  • genomic profiles e.g., of mRNAs, protein-DNA binding, protein levels, protein phosphorylation, etc.
  • genomic profiles e.g., of mRNAs, protein-DNA binding, protein levels, protein phosphorylation, etc.
  • the invention adapts CRISPR-Cas9 technology to develop a marker based genome-wide knockout screen in primary mammalian cells derived from Cas9-expressing transgenic mice, and apply it to dissect gene regulator ⁇ ' circuits in the response of dendritic cells ("DCs") to lipopolysaccharide (“LPS,”)
  • DCs dendritic cells
  • LPS lipopolysaccharide
  • aspects of the present invention may address herein discussed challenges and need in the art by advantageously providing methods, systems, compositions and models for ex vivo and in vivo modeling of processes involving leukocytes, by introducing mutations into leukocytes, including in specific subsets thereof, for instance in order to modulate leukocyte responses such as e.g. activation or d fferentiation or to identify genes associated with such responses.
  • the invention provides a method for modulating leukocyte activity, comprising delivering to a leukocyte a vector containing nucleic acid molecule(s), whereby the leukocyte contains Cas9 and the integrated vector expresses one or more RNAs to guide the Cas9 to introduce mutations in one or more target genetic loci in the leukocyte, thereby modulating expression of one or more genes in the leukocyte,
  • the invention provides a method of identifying a gene associated with a leukocyte response, comprising (a) delivering to a plurality of leukocytes a plurality of different vectors containing nucleic acid molecule(s), whereby the leukocytes contain Cas9 and each vector expresses one or more RNAs to guide the Cas9 to introduce mutations in different target genetic loci in the leukocytes, thereby modulating expression of different genes expressed in the leukocytes; (b) measuring a response in the leukocytes; (c) isolating leukocyte(s) in which the response is modulated following delivery of the vector; and (d) thereby identifying gene(s) associated with the leukocyte response,
  • the invention provides a method for modeling an aberrant leukocyte response or a disease associated with leukocytes, comprising delivering to leukocytes a vector containing nucleic acid molecule(s), whereby the leukocytes contain Cas9 and the integrated or non-integrated vector expresses one or more RNAs to guide the Cas9 to introduce mutations in one or more target genetic loci in the leukocytes, thereby modulating expression of one or more genes expressed in the leukocytes, and wherein expression of the one or more genes is associated with the response or disease.
  • the invention provides an individualized or personalized treatment of a leukocyte-associated disease in a subject in need of such treatment comprising (a) delivering to leukocytes ex vivo a vector containing nucleic acid molecule(s), whereby the leukocytes express Cas9 and the vector expresses one or more RNAs to guide the Cas9 to introduce mutations in one or more target genetic loci in the leukocytes, thereby modulating expression of one or more genes expressed in the leukocytes, and wherein expression of the one or more genes is correlated to the disease; (b) testing treatment(s) for the disease on the leukocytes to which the vector has been delivered that comprise mutations in genes correlated to the disease; and (c) treating the subject based on results from the testing of treatments ) of step (b).
  • the invention provides an isolated leukocyte comprising a vector containing a nucleic acid molecule(s), wherein the leukocyte contains or conditionally or inducibly expresses Cas9 and the vector encodes one or more RNAs capable of guiding the Cas9 to introduce mutations in one or more target genetic loci in the leukocyte, and thereby modulating expression of one or more genes expressed in the leukocyte.
  • the invention provides an isolated leukocyte comprising a vector containing a nucleic acid molecule(s), wherein the leukocyte contains or conditional!)' or inducibly expresses Cas9, one or more target genetic loci in the leukocyte comprise mutations derived from Cas9 activity guided by one or more RNAs encoded by the vector, and the mutations modulate expression of one or more genes expressed in the leukocyte.
  • aspects of the invention provide methods for enabling rapid and direct in vivo and ex vivo modeling of the dynamics of leukocyte responses (e.g. activation and differentiation) associated with mutations in leukocytes.
  • aspects of the present invention involve sequence targeting, such as genome perturbation or induction of multiple mutations using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system or components thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the invention provides systematic reverse engineering of causal genetic variations, including through selective perturbation of individual, and moreover, multiple genetic elements.
  • non-human eukaryote e.g., animal, such as fish, e.g., zebra fish, mammal, e.g., primate, e.g., ape, chimpanzee, macaque, rodent, e.g., mouse, rabbit, rat, canine or dog, livestock (cow bovine, sheep/ovine, goat or pig), fowl or poultry, e.g., chicken, models that constitutively or through induction or through administration or delivery, have cells that contain Cas9.
  • animal such as fish, e.g., zebra fish, mammal, e.g., primate, e.g., ape, chimpanzee, macaque, rodent, e.g., mouse, rabbit, rat, canine or dog, livestock (cow bovine, sheep/ovine, goat or pig), fowl or poultry, e.g., chicken, models that constitutively or through induction or through
  • the invention provides tools for studying genetic interaction between multiple individual genetic elements by allowing selective perturbation of e.g., one or more gene(s)/genetic element(s) in leukocytes or a subset thereof.
  • the invention provides methods for using one or more elements/components of a CRISPR-Cas system via a vector and/or particle and/or nanoparticle delivery formulation or system as a means to modify a target polynucleotide in leukocytes.
  • the delivery is via a viral vector (e.g., AAV, adenovirus, lentivirus, preferably a lentivirus) or a cell-penetrating peptide (CPP) (e.g., a peptide of about 8-13 amino acids, typically about 11 amino acids, having an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids— polycationic or amphipathic, respectively).
  • a viral vector e.g., AAV, adenovirus, lentivirus, preferably a lentivirus
  • CPP cell-penetrating peptide
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide in leukocytes or a subset thereof.
  • the CRISPR complex of the invention has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in leukocytes or a subset thereof, e.g. in dendritic cells, monocytes, macrophages, T cells, B cells, natural killer (NK) T cells, NK cells, neutrophils, eosinophils, basophils, mast cells, innate lymphoid cells, follicular dendritic cells, stellate cells as well as precursors of mature leukocytes such as hematopoietic stem and progenitor cells.
  • modifying e.g., deleting, inserting, translocating, inactivating, activating
  • the modification may occur ex vivo or in vitro in leukocytes derived from a non-human transgenic Cas9 organism, for instance in a cell culture and in some instances not in vivo.
  • the modification occurs ex vivo in primary cells, e.g. in primary leukocytes extracted from a human or non-human animal or in cells derived from primary leukocytes by differentiation ex vivo. In other embodiments, it may occur in vivo.
  • leukocytes are modified ex vivo and then reintroduced into a different individual human or non-human animal, e.g. via adoptive transfer.
  • the invention provides a method of modifying an organism or a non- human organism, or leukocytes derived from an organism or non-human organism, by- manipulation of a target sequence in a genomic locus of interest comprising: Delivering, e.g., via pneumoniae(s) or nanoparticle(s) or vector(s) (e.g., viral vector, e.g., AAV, adenovirus, Ientivirus) a non-naturally occurring or engineered composition into leukocytes of or from the organism.
  • Delivering e.g., via a particle(s) or vector(s) (e.g., viral vector, e.g., AAV, adenovirus, Ientivirus) a non-naturally occurring or engineered composition into leukocytes of or from the organism.
  • the composition can comprise: A) L RNA(s) having polynucleotide sequence(s), e.g., a CRISPR-Cas system chimeric RNA (chiRNA) having a polynucleotide sequence, wherein the polynucleotide sequence comprises: (a) a guide sequence capable of hybridizing to a target sequence in a leukocyte, (b) a tracr mate sequence, and (c) a tracr sequence; wherein (a), (b) and (c) are arranged in a 5' to 3' orientation.
  • the composition can also comprise A) II.
  • a polynucleotide sequence encoding a CRISPR enzyme advantageously comprising at least one or more or two or more nuclear localization sequences.
  • the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (I) the guide sequence that is hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridizable to the tracr sequence.
  • the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA.
  • Cas9 is already present in the leukocyte, e.g., through the leukocyte having already been provided (A) II. or through the leukocyte expressing Cas9, e.g., through the leukocyte having been transformed to express Cas9, e.g., Cas9 is expressed constitutively or conditionally or inducibly— for instance when the leukocyte is part, of or from a non-human transgenic e karyote, e.g., animal, mammal, primate, rodent, etc as herein discussed— then (A) L is provided as the CRISPR complex is formed in situ or in vivo.
  • a non-human transgenic e karyote e.g., animal, mammal, primate, rodent, etc as herein discussed
  • the invention provides a method of modifying an organism or a non-human organism, or leukcoytes derived from an organism or non-human organism, by manipulation of a target sequence in a genomic locus of interest comprising: Delivering, e.g., via particle(s) or nanoparticle(s) or vector(s) (e.g., viral vector, e.g., AAV, adenovirus, Ientivirus) a non-naturally occurring or engineered composition into leukocytes of or from the organism.
  • the composition can comprise (B) I.
  • polynucleotides comprising: (a) a guide sequence capable of hybridizing to a target sequence in a leukocyte, and (b) at least one or more tracr mate sequences.
  • the composition can also comprise (B) II. a polynucleotide sequence comprising a tracr sequence.
  • the composition can also comprise (B) III. a polynucleotide sequence encoding a CRISPR enzyme advantageously comprising at least one or more or two or more nuclear localization sequences.
  • the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence.
  • the CRISPR complex comprises the CRISPR.
  • the Cas9 is already present in the leukocyte, e.g., through the leukocyte having already been provided (B) III.
  • (B) I. and (B) II. are provided as the CRISPR complex is formed in situ or in vivo.
  • components I and II or I, II and III or the foregoing embodiments can be delivered separately; for instance, in embodiments involving components ⁇ , II and III, components I and II can be delivered together, while component III can be delivered separately, e.g., prior to components I and II, so that the leukocyte expresses Cas9.
  • the invention comprehends delivering a CRISPR enzyme comprising delivering to a leukocyte mRNA encoding the CRISPR. enzyme, e.g., via nanoparticle complex(es).
  • the CRISPR enzyme is a Cas9.
  • the Cas9 enzyme is constitutively present, e.g., through knock-in.
  • the Cas9 enzyme is constitutively present in vivo (e.g, a non-human transgenic eukaryote, animal, mammal, primate, rodent, etc) or ex vivo (comprising a vector containing nucleic acid molecule(s) for in vivo expression of the Cas9).
  • the CRISPR enzyme is a type I or III CRISPR enzyme, preferably a type II CRISPR enzyme. This type II CRISPR. enzyme may be any Cas enzyme.
  • a preferred Cas enzyme may be identified as Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with, multiple nuclease domains from the type II CRISPR system.
  • the Cas9 enzyme is from, or is derived from, SpCas9 or SaCas9.
  • SpCas9 or SaCas9 are those from or derived from S. pyogenes or S. aureus Cas9.
  • derived Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein.
  • the terms Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent.
  • the Cas enzyme can be for instance any naturally-occurring bacterial Cas9 as well as any chimaeras, mutants, homologs or orthologs. Many of the residue numberings used herein refer to the Cas9 enzyme from the type I I CRISPR locus in Streptococcus pyogenes (annotated alternatively as SpCas9 or spCas9). However, it will be appreciated that this invention includes many more Cas9s from other species of microbes, e.g., orthologs of SpCas9, or Cas9s derived from microbes in addition to S. pyogenes, e.g., SaCas9 derived from S.
  • Cas9 orthologs typically share the genera! organization of 3-4 RuvC domains and a HNH domain. The 5' most RuvC domain cleaves the non-complementary strand, and the HNH domain cleaves the complementary strand.
  • the catalytic residue in the 5' RuvC domain is identified through homology comparison of the Cas9 of interest with other Cas9 orthologs (from S. pyogenes type II CRISPR. locus, S. thermophilics CRISPR locus 1, S, thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPR locus), and the conserved Asp residue (D10) is mutated to alanine to convert Cas9 into a complementary-strand, nicking enzyme. Similarly, the conserved His and Asn residues in the HNH domains are mutated to Alanine to convert Cas9 into a non-complementary-strand nicking enzyme.
  • both sets of mutations may be made, to convert Cas9 into a non-cutting enzyme (i.e. lacking nuclease activity).
  • the Cas9 may comprise one or more mutations and may be used as a generic D A binding protein with or without fusion to a functional domain, e.g. a domain which activates or inhibits gene expression .
  • the Cas9 may be a mutant Cas9 lacking nuclease activity that activates or inhibits expression of a target gene guided by the RNA expressed by the vector.
  • the mutations may be artificially introduced mutations or gain- or loss-of- function mutations.
  • the mutations may include but are not limited to mutations in one of the catalytic domains (e.g., D10 and H840) in the RuvC and HNH catalytic domains respectively; or the CRISPR enzyme can comprise one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A or D986A and/or one or more mutations in a RuvCl or HNH domain of the CRISPR enzyme or has a mutation as otherwise as discussed herein, in one aspect of the invention, the Cas9 enzyme may be fused to a protein, e.g., a TAG, and/or an inducible/controllahle domain such as a chemically mducible/control!able domain.
  • a protein e.g., a TAG
  • an inducible/controllahle domain such as a chemically mducible/control!able domain.
  • the Cas9 in the invention may be a chimeric Cas9 proteins; e.g., a Cas9 having enhanced function by being a chimera.
  • Chimeric Cas9 proteins may be new Cas9 containing fragments from more than one naturally occurring Cas9. These may comprise fusions of N-terminal fragment(s) of one Cas9 homolog with C-temnnai fragment(s) of another Cas9 homolog.
  • the Cas9 can be delivered into the leukocyte in the form of mRNA.
  • the expression of Cas9 can be under the control of an inducible promoter.
  • the tracrRNA and direct repeat sequences can be mutant sequences or the invention can encompass RNA of the CRISPR-Cas system that includes mutant chimeric guide sequences that allow for enhancing performance of these R As in leukocytes.
  • a suitable promoter such as the Pol III promoter, such as a U6 promoter, can be added onto the guide RNA that is advantageously delivered via AAV or particle or nanoparticle.
  • aspects of the invention also relate to the guide RNA being transcribed in vitro or ordered from a synthesis company and directly transfected. Expression of RNA(s), e.g., guide RNAs or sgRNA.
  • the leukocyte under the control of the T7 promoter driven by the expression of T7 polymerase in the leukocyte is also envisioned, in an advantageous embodiment, the leukocyte is a human leukocyte. In another preferred embodiment, the leukocyte is a non-human mammalian, e.g., primate, rodent, e.g., mouse, rat, rabbit.
  • rodent e.g., mouse, rat, rabbit.
  • the leukocyte is a patient-specific leukocyte, e.g., a leukocyte in which one or more, e.g., a plurality, such as 3 or more, e.g., 3-50 or more, mutations associated or correlated with a patient's genetic disease, e.g., a leukocyte-associated disease, are expressed in the leukocyte, e.g., via Cas9 being present in the leukocyte and RNA(s) for such mutations delivered to the leukocyte (e.g., any whole number between 1 and 50, e.g., between 2 and 50, such as between 3 and 50 of mutations, with it noted that in some embodimen ts there can be up to 16 different RNA(s), e.g., sgRNAs, e.g., each having its own a promoter, in a vector, such as a lentiviral vector, and that when each sgRNA does not have its own promoter, there can
  • a codon optimized sequence can be a sequence optimized for a eukaryote, or for specific cells such as leukocytes. It will be appreciated that where reference is made to a polynucleotide, where that polynucleotide is RNA and is said to 'comprise' a feature such as a tracr mate sequence, the RNA sequence includes the feature. Where the polynucleotide is DNA and is said to comprise a feature such as a tracr mate sequence, the DNA sequence is or can be transcribed into the RNA that comprises the feature at issue.
  • the DNA or RNA sequence referred to is, or can be, translated (and in the case of DNA transcribed first).
  • an RNA encoding the CRISPR enzyme is provided to a leukocyte, it is understood that the RNA is capable of being translated by the leukocyte into which it is delivered.
  • manipulation of a target sequence Applicants mean the alteration of the target sequence, which may include the epigenetic manipulation of a target sequence. This epigenetic manipulation may be of the chromatin state of a target sequence, such as by modification of the methylation state of the target sequence (i.e.
  • the invention in some embodiments comprehends a method of modifying a eukaryote, such as a Cas9 transgenic eukaryote comprising delivering, e.g., via vector(s) and/or particle(s) and/or nanoparticles a non- naturally occurring or engineered composition.
  • the composition comprises: I.
  • a first regulatory element operably linked to (a) a first guide sequence capable of hybridizing to a first target sequence, and (b) at least one or more tracr mate sequences
  • II. a second regulatory element operably linked to (a) a second guide sequence capable of hybridizing to a second target sequence, and (b) at least one or more tracr mate sequences.
  • III. a third regulatory element operably linked to (a) a third guide sequence capable of hybridizing to a third target sequence, and (b) at least one or more tracr mate sequences
  • the composition can involve V.
  • a fifth regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme (e.g., for establishing the Cas9 transgenic eukaryote).
  • Components I, ⁇ , 111 and IV are located on the same or different vectors and/or particles and/or nanopardcles of the system.
  • the tracr mate sequence hybridizes to the tracr sequence and the first, second and the third guide sequences direct sequence-specific binding of a first, second and third CRISPR.
  • the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridizable to the first target sequence, and (2) the tracr mate sequence that is hybridizable to the tracr sequence
  • the second CRISPR complex comprises the CRISPR enzyme complexed with (I) the second guide sequence that is hybridizable to the second target sequence, and (2) the tracr mate sequence that is hybridizable to the tracr sequence
  • the third CRISPR complex comprises the CRISPR enzyme complexed with (1) the third guide sequence that is hybridizable to the third target sequence, and (2) the tracr mate sequence that is hybridizable to the tracr sequence, whereby in a Cas9 transgenic eukaryote or leukocyte thereof, at least three (3) mutations may be induced, and advantageously the mutations are correlated or associated with an aberrant leukocyte response or a leukocyte-associated disease condition, whereby
  • the invention also provides a vector system as described herein.
  • the system may comprise one, two, three or four different vectors; and the system may comprise one, two, three or four different nanoparticie complex(es) deliverying the components) of the system.
  • Components I, II, III and IV may thus be located on one, two, three or four different vectors, and may be delivered by one, two, three or four different particle or nanoparticie complex(es) or AAVs or components I, II, III and IV can be located on same or different vector(s) / particle(s) / ' nanoparticie(s), with all combinations of locations envisaged. And complexes that target leukocytes or subsets thereof are advantageous.
  • any or ail of the polynucleotide sequence encoding the CR.1SPR. enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence is/are RNA; and advantageously delivered via nanoparticle eomplex(es) or a cell-penetrating peptide (CPP).
  • RNAs comprising a guide sequence fused to a tracr mate sequence and a tracr sequence.
  • the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell, e.g., a primate or rodent cell such as a mouse cell or a human cell (e.g. a mammalian, mouse or human leukocyte).
  • the invention also comprehends an engineered, non-natttrally occurring vector system.
  • the system comprises one or more vectors comprising: (a) a first regulatory element operabiy linked to each of two or more e.g. three, CRISPR-Cas system guide RNAs that target a first strand, a second strand and a third strand respectively of a double stranded DNA molecule.
  • the system can also comprise (b) a second regulatory element operabiy linked to a Cas protein.
  • Components (a) and (b) are located on same or different vectors of the system, but advantageously separate vectors as it is preferred that leukocytes receiving (a) contain Cas9, e.g., via the leukocytes being those of a transgenic Cas9 eukaryote (whereby (b) may have been administered to cells that gave rise to the eukaryote).
  • the guide RNAs target DNA and at least three mutations are induced in the leukocytes, e.g., mutations correlated to or associated wit an aberrant leukocyte response or a disease associated with leukocytes, such as an immune system disorder or cancer.
  • the invention provides a method of modifying a target polynucleotide in a leukocyte, for instance in order to modulate leukocyte activity.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme compiexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence; and advantageously the complex or a component thereof has been delivered via nanoparticle complex(es).
  • said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, said cleavage results in increased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide.
  • the mutation can be a mutation correlated to or associated with an aberrant leukocyte response or a leukocyte-associated disease condition, such as an immune system disorder.
  • said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
  • said mutation results in one or more changes in promoter or enhancer sites that modulate gene expression.
  • said mutation results in one or more changes in splice sites that modulate splicing of the gene.
  • vectors are delivered to the leukocyte in a transgenic Cas9 eukaryote.
  • said modifying takes place in said leukocyte in a cell culture, preferably a primary cell culture.
  • the invention provides a method of generating a model leukocyte or a model Cas9 transgenic eukaryote comprising mutated gene(s) in leukocytes, e.g., having one or more, e.g., a plurality, e.g., 3 or more, such as 3-50 or more mutations correlated to or associated with an aberrant leukocyte response or a leukocyte-associated disease such as an immune system disorder (e.g., any whole number between 1 and 50, e.g., between 2 and 50, such as between 3 and 50 of mutations, with it noted that in some embodiments there can be up to 16 different RNA(s), e.g., sgRNAs each having its own a promoter, in a vector, such as AAV, and that when each sgRNA does not have its own promoter, there can be twice to thrice that amount of different RNA(s), e.g., sgRNAs, e.g., 32
  • a target polynucleotide can be inactivated to effect the modification of the expression in a leukocyte. For example, upon the binding of a CRISPR complex to a target sequence in a leukocyte, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild- type sequence does. For example, a protein or microRNA coding sequence may be inactivated such that the protein or microRNA or pre -microRNA transcript is not produced.
  • the target sequence is flanked or followed, at its 3' end, by a PAM (protospacer- adjacent motif) suitable for the CRISPR enzyme, typically a Cas and in particular a Cas9.
  • a PAM protospacer- adjacent motif
  • a suitable PAM is 5'-NRG or 5 -NNGRR for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively.
  • Delivery can be in the form of a vector which may be a plasmid or other nucleic acid molecule form, especially when the delivery is via a nanoparticle complex; and the vector also can be viral vector, such as a herpes, e.g., herpes simplex virus, lenti- or baculo- or adeno-viral or adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, micro vesicles, gene guns/means of attaching vectors to gold nanoparticles and cell -penetrating peptides (CPP)) and are provided, especially as to those aspects of the complex not delivered via a nanoparticle complex.
  • a herpes e.g., herpes simplex virus, lenti- or baculo- or adeno-viral or adeno-associated viral vectors
  • other means of delivery are known (such as yeast systems, micro vesicles, gene guns/means of attaching vector
  • a vector may mean not only a viral or yeast system (for instance, where the nucleic acids of interest may be operably linked to and under the control of (in terms of expression, such as to ultimately provide a processed RNA) a promoter), but also direct delivery of nucleic acids into a host cell; and advantageously the complex or a component thereof is delivered via nanoparticle complex(es). Also envisaged is a method of delivering the present CR ISPR enzyme comprising delivering to a leukocyte mRNA encoding the CRISPR enzyme; and advantageously the complex or a component thereof has been delivered via nanoparticle complex(es).
  • the CRISPR enzyme is truncated, and/or comprised of less than one thousand amino acids or less than four thousand amino acids, and/or is a nuclease or nickase, and/or is codon-opti ized, and/or comprises one or more mutations, and/or comprises a chimeric CRISPR enzyme, and/or the other options as herein discussed.
  • lenti virus is a preferred vector.
  • RNA(s) or guide R As or sgR As formulated in one or more delivery vehicles e.g., where some guide RNAs are provided in a vector and others are formulated in nanoparticles; and these may be provided alone (e.g., when Cas9 is already in a leukocyte) or with a Cas9 delivery system.
  • the Cas9 is also delivered in a nanoparticle formulation.
  • the RNA(s) or guide RNA or sgRNA-vector and/or particle and/or nanoparticle formulation(s) and the Cas9 vector and/or particle and/or nanoparticle formulation(s) may be delivered separately or may be delivered substantially contemporaneously (i.e., co-delivery).
  • Sequential delivery could be done at separate points in time, separated by days, weeks or even months.
  • sequential delivery can include initially administering or delivering the Cas9 vector and/or particle and/or nanoparticle formulation(s) to cells that give rise to the non-human Cas9 transgenic eukaryote, and thereafter, at a suitable time in the life of the transgenic eukaryote, administering the RNA(s) or guide RNA or sgRNA- vector and/or particle and/or nanoparticle formuIation(s), e.g., so as to give rise to one or more, advantageously e.g., a plurality, e.g., 3 or more, advantageously 3-50 or more mutations in leukocytes in or derived from the transgenic eukaryote (e.g.
  • the vector may comprise at least 3 sgRNAs, at least 8 sgRNAs, at least 16 sgRNAs, at least 32 sgRNAs, at least 48 sgRNAs, or at least 50 sgRNAs.
  • the vector may comprise 1 -2 sgRNAs, 1-3 sgRNAs, 1-4 sgRNAs, 1-5 sgRNAs, 3-6 sgRNAs, 3-7 sgRNAs, 3-8 sgRNAs, 3-9 sgRNAs, 3-10 sgRNAs, 3-16 sgRNAs, 3-30 sgRNAs, 3-32 sgRNAs, 3-48 sgRNAs or 3-50 sgRNAs.
  • vector e.g., AAV, adenovirus, lentivirus
  • particle and/or nanoparticle formulations comprising one or more RNA(s) e.g.
  • guide RNAs or sgRNA are adapted for delivery in vitro, ex vivo or in vivo in the context of the CRISPR-Cas system, e.g., so as to form CRISPR-Cas complexes in vitro, ex vivo or in vivo, to different target genes in leukocytes.
  • Multiplexed gene targeting using nanoparticle formulations comprising one or more guide RNAs are also envisioned.
  • a nanoparticle formulation comprising one or more components of the CRISPR-Cas system is provided.
  • a RNA(s) or gRNA or sgRNA-nano article formulation comprising one or more guide RNAs or sgRNA is provided.
  • a composition comprising a nanoparticle formulation comprising one or more components of the CRISPR-Cas system.
  • a composition e.g., a pharmaceutical or veterinary composition, comprising a vector (e.g., AAV, adenovirus, lentivirus) and/or particle and/or nanoparticle formulation comprising one or more components of the CRISPR-Cas system and/or nucleic acid molecule(s) coding therefor, advantageously with such nucleic acid moleeule(s) operably linked to promoter(s) is provided. Accordingly, in certain embodiments, it may be useful to deliver the RNA(s) or guide RNA.
  • sgRNA e.g., vector and/or particle and/or nanoparticle formulations separately from the Cas9 or nucleic acid molecule(s) coding therefor.
  • a dual-delivery system is envisaged such that the Cas 9 may be delivered via a vector and the RNA(s), e.g., guide R ' NAs or sgRNA are / ' is provided in a particle or nanoparticle formulation, for example, first Cas9 vector is delivered via a vector system followed by delivery of sgRNA-nanoparticle formulation.
  • Vectors may be considered in the broadest light as simply any means of delivery, rather than specifically viral vectors.
  • the present invention provides a Cas9 transgenic eukaryote, e.g., mouse.
  • the Cas 9 transgenic eukaryote, e.g., mouse comprises a Cas9 transgene knocked into the Rosa26 locus
  • the present invention provides a Cas9 transgenic eukaryote, e.g., mouse wherein Cas9 transgene is driven by the ubiquitous CAG promoter thereby providing for constitutive expression of Cas9 in all tissues/cells/ceil types of the mouse.
  • the present invention provides a Cas9 transgenic eukaryote, e.g., mouse wherein the Cas9 transgene driven by the ubiquitous CAG promoter further comprises a Lox- Stop-polyA-Lox (LSL) cassette (Rosa26-LSL-Cas9 mouse) thereby rendering Cas9 expression inducible by the Cre recombinase.
  • LSL Lox- Stop-polyA-Lox
  • the present invention provides a constitutive Cas9 expressing eukaryote, e.g., mouse line obtained by crossing of the Rosa26-LSL-Cas9 mouse with a beta-actin-Cre eukaryote, e.g., mouse line.
  • progeny(ies) derived from said Cas9 expressing eukaryote may be successfully bred over at least five generations without exhibiting increased levels of genome instability or cellular toxicity.
  • the present invention provides a modular viral vector contract comprising a plurality of sgRNAs driven by a single RNA polymerase III promoter (e.g., U6), wherein the sgRNA s are in tandem, or where each of the sgRNAs in driven by one RNA polymerase III promoter.
  • the present invention provides a modular viral vector construct comprising one or more cassettes expressing Cre recombinase, a plurality of sgRNAs to guide Cas9 cutting, and a HDR template to model the dynamics of a complex pathological disease or disorder involving two or more genetic elements simultaneously using a single vector construct.
  • the complex pathological disease or disorder is a disease associated with leukocytes, e.g. an immune system disorder or cancer. It can be appreciated that any kind of disease associated with leukocytes is within the scope of the present invention.
  • the present invention provides for modeling of immune system disorders
  • the present invention provides a modular viral vector construct to model the dynamics of multiple gene mutations in leukocytes simultaneously using a single vector.
  • the modular viral vector construct comprises one or more cassettes expressing Cre recombinase, a plurality of sgRNAs to guide Cas9 cutting, and optionally including a Homology Directed Repair (HDR) template to introduce specific gain-of-function mutations or precise sequence substitution in target loci.
  • HDR Homology Directed Repair
  • the present invention provides a method for simultaneously introducing one or more mutations ex vivo in leukocytes, or in vivo in the same animal comprising delivering a single viral vector construct, wherein the viral vector construct comprises one or more cassettes expressing Cre recombinase, a plurality of sgRNAs to guide Cas9 cutting, and a HDR template for achieving targeted insertion or precise sequence substitution at specific target loci of interest.
  • the present invention provides a method for generating loss-of-function mutation or mutations and/or gain-of-function mutation or mutations.
  • the present invention provides a method for delivering ex vivo or in vivo of any of the constructs disclosed herein into leukocytes.
  • AAV AAV is used selection of the AAV serotype is based on its suitability and specificity for a tissue type.
  • a lentiviral vector is used for delivery to leukocytes
  • the present invention provides a method for ex vivo and/or in vivo genome editing comprising delivering any of the above modular viral vector constructs, which comprise one or more cassettes expressing Cre recombinase, a plurality of sgRNAs to guide Cas9 cutting, and optionally one or more HDR template(s), into a Cas9 transgenic mouse (e.g., Rosa26-LSL-Cas9) or into leukocytes from a Cas9 transgenic mouse (e.g., Rosa26-LSL-Cas9).
  • the viral vector is lenti virus.
  • the present invention provides a Cas9 transgenic non-human eukaryote, or leukocytes derived therefrom, e.g., an animal or cellular model for an aberrant leukocyte response or a disease associated with leukocytes, e.g. an immune system disorder or cancer, said model having loss-of-function mutations and/or gain-of- function mutation in leukocytes in one or more genes associated with the response or disease.
  • a Cas9 transgenic non-human eukaryote, or leukocytes derived therefrom e.g., an animal or cellular model for an aberrant leukocyte response or a disease associated with leukocytes, e.g. an immune system disorder or cancer, said model having loss-of-function mutations and/or gain-of- function mutation in leukocytes in one or more genes associated with the response or disease.
  • a Cas9 transgenic non-human eukaryote and leukocytes derived therefrom e.g., an animal or cellular model with multiple mutations in any number of loci in leukocytes can be envisioned and are within the scope of the present invention.
  • transgenic non-human eukaryote or leukocytes derived therefrom provides a valuable tool for research purposes, e.g., to delineate specific roles/contribution of individual genes expressed in leukocytes in leukocyte responses and to progression of a disease associated with leukocytes, to recapitulate specific combinations of mutations in a given leukocyte type, and opens the door for developing and testing new therapeutic interventions for diseases associated with leukocytes involving mutations at one or multiple loci.
  • Such uses are within the scope of the present invention.
  • the present invention provides a method of treating or inhibiting the development of a disease associated with leukocytes in a subject in need thereof, comprising providing individualized or personalized treatment (or an individualized or personalized model or patient specific-modeling) comprising: delivering RNA(s), e.g., sgRNA, that targets a genetic locus correlated or associated with the disease, e.g.
  • RNA(s) e.g., sgRNA
  • a Cas9 non-human transgenic eukaryote e.g., animal, mammal, primate, rodent, fish etc as herein discussed
  • leukocytes derived therefrom e.g., via vector such as AAV, adenovirus, lentivirus, or particle(s) or nanoparticle(s), whereby mutation(s), advantageously a plurality, e.g., 1 or more, e.g., a plurality, such as 3 or more or 3-50 or more mutations (e.g., any whole number between 3 and 50 or more of mutations, with it noted that in some embodiments there can be up to 16 different RNA(s), e.g., sgRNAs each having its own a promoter, in a vector, such as AAV, and that when each sgRNA does not have its own promoter, there can be twice to thrice that amount of different RNA(s), e.g., sgRNAs
  • the obtaining and/or extrapolating data can be subjecting the eukaryote or leukocytes derived therefrom to putative treatment(s) and/or therapy(ies), e.g., gene therapy, ascertaining whether such putative treatment(s) and/or therapy(ies) give rise to remission or treatment or al leviation or remission of the disease or to a process associated with disease remission (e.g.
  • the invention thus allows for one to ascertain whether a particular treatment and/or therapy may be effective as to a particular individual's disease.
  • the invention provides vector(s), particle(s) or nanoparticle(s) containing nucleic acid molecule(s), whereby in vivo in a eukaryotic ceil containing or conditionally or inducibly expressing Cas9: the vector! s) express!
  • RNAs es
  • a plurality of RNAs to guide the Cas9 and optionally delivers donor templates (e.g., HDR templates, and in certain embodiments advantageously includes and delivers such donor templates), and optionally in the event Cas9 is conditionally or inducibly expressed in the cell that which induces Cas9, e.g., Cre recombinase; whereby a plurality of specific mutations or precise sequence substitutions in a plurality of target loci are introduced.
  • the vector(s) can be a viral vector such as lentivims, adenovirus, or adeno-associated virus (AAV), e.g., AAV6 or AAV9.
  • the Cas9 can be from S. thermophilics, S. aureus, or S.
  • the eukaryotic cell e.g. leukocyte
  • the eukaryotic cell can comprise a Cas9 transgene flmctionally or operatively linked to a constitutive promoter, or a tissue specific promoter, or an inducible promoter; and, the eukaryotic cell (e.g., leukocyte) can be part of a non-human transgenic eukaryote, e.g., a non-human mammal, primate, rodent, mouse, rat, rabbit, canine, dog, cow, bovine, sheep, ovine, goat, pig, fowl, poultry, chicken, fish; advantageously a mouse.
  • a non-human transgenic eukaryote e.g., a non-human mammal, primate, rodent, mouse, rat, rabbit, canine, dog, cow, bovine, sheep, ovine, goat, pig, fowl, poultry, chicken, fish; advantageously a mouse.
  • the leukocyte can be an isolated eukaryotic cell, e.g., from any of the foregoing non- human animals or from a human or the leukocyte can be part of a non-human transgenic animal as herein discussed.
  • the leukocyte or non-human transgenic animal can express an additional protein or enzyme, such as Cre; and, the expression of Cre can be driven by coding therefor flmctionally or operatively linked to a constitutive promoter, or a tissue specific promoter, or an inducible promoter.
  • the RNAs to guide Cas9 can comprise CRISPR RNA and transact! vating (tracr) RNA.
  • the RNAs to guide Cas9 can comprise chimeric single guide RNA (sgRNA).
  • sgRNA chimeric single guide RNA
  • the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned can be about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence can be about or more than about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%).
  • a guide or RNA or sgRNA can be about or more than about 5, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • a guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length,
  • the veetor(s) can include the regulatory element(s), e.g., promoter(s).
  • the vector(s) can comprise at least 3 or 8 or 16 or 32 or 48 or 50 RNA(s) (e.g., sgRNAs), such as 1-2, 1-3, 1-4 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s) (e.g., sgRNAs).
  • a promoter for each RNA there can be a promoter for each RNA (e.g., sgRNA), advantageously when there are up to about 16 R_NA(s) (e.g., sgRNAs); and, when a single vector provides for more than 16 RNA(s) (e.g., sgRNAs), one or more promoter(s) can drive expression of more than one of the RNA(s) (e.g., sgRNAs), e.g., when there are 32 RNA(s) (e.g., sgRNAs), each promoter can drive expression of two RNA(s) (e.g., sgRNAs), and when there are 48 RNA(s) (e.g., sgRNAs), each promoter can drive expression of three RNA(s) (e.g., sgRNAs).
  • RNA(s) e.g., sgRNA(s) for a suitable exemplary vector such as lenti virus or AAV
  • a suitable promoter such as the U6 promoter, e.g., U6-sgRNAs.
  • the packaging limit of AAV is ⁇ 4.7 kb.
  • the length of a single U6-sgRNA (plus restriction sites for cloning) is 361 bp. Therefore, the skilled person can readily fit about 12-16, e.g., 13 U6-sgRNA cassettes in a single vector.
  • the skilled person can also use a tandem guide strategy to increase the number of U6-sgRNAs by approximately 1.5 times, e.g., to increase from 12-16, e.g., 13 to approximately 18-24, e.g., about 19 U6-sgRNAs. Therefore, one skilled in the art can readily reach approximately 18-24, e.g., about 19 promoter-RNAs, e.g., U6-sgRNAs in a single vector, e.g., an AAV or lentivirus vector.
  • a further means for increasing the number of promoters and RNAs, e.g., sgRNA(s) in a vector is to use a single promoter (e.g., U6) to express an array of RNAs, e.g., sgRNAs separated by cleavabie sequences.
  • a single promoter e.g., U6
  • promoter-RNAs e.g., sgRNAs in a vector
  • express an array of promoter-RNAs e.g., sgRNAs separated by cleavabie sequences in the intron of a coding sequence or gene; and, in this instance it is advantageous to use a polymerase II promoter, which can have increased expression and enable the transcription of long RNA in a tissue specific manner.
  • AAV or lentivirus may package U6 tandem sgRNA targeting up to about 50 genes.
  • veetor(s) e.g., a single vector, expressing multiple RNAs or guides or sgRNAs under the control or operatively or functionally linked to one or more promoters— especially as to the numbers of RNAs or guides or sgRNAs discussed herein, without any undue experimentation.
  • RNA(s) can be functionally or operatively linked to regulatory element(s) and hence the regulatory element(s) drive expression.
  • the promoters can be constitutive promoters) and/or inducible promoter(s) and/or tissue specific promoters).
  • the promoter can be selected from the group consisting of RNA polymerases, pol 1, pol II, pol 111, T7, U6, HI, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PG ) promoter, and the EF la promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter the dihydrofolate reductase promoter
  • ⁇ -actin promoter the phosphoglycerol kinase (PG ) promoter
  • PG phosphoglycerol kinase
  • each RNA is specific to a different target sequence.
  • Each different target sequence can be associated with or correlated to a form of disease associated with or correlated to or related to leukocytes or white blood cells or a leukocyte response or associated with an immune system disorder or with a cancer or with the response of leukocytes to cancer.
  • Each RNA or sgRNA can be specific to a different target sequence but these RNA(s) or sgRNA(s) target specific gene sequences associated with or correlated to a disease of leukocytes or white blood cells or a leukocyte response or associated with an immune system disorder or with a cancer or with the response of leukocytes to cancer, advantageously a particular type or form of disease of leukocytes or white blood cells or a leukocyte response or associated with an immune system disorder or with a cancer or with the response of leukocytes to cancer; for instance each RNA or sgRNA can be specific to a different target sequence but the target sequences of the RNA(s) or sgRNA(s) are associated with or correlated to the same type or form of disease associated with or correlated to or related to leukocytes or white blood cells or a leukocyte response or associated with an immune system disorder or with a cancer or with the response of leukocytes to cancer; such as leukopenia, or leukemia (e.g.
  • each sgRNA can be driven by an independent promoter, e.g., U6 promoter.
  • the vector can be a !entivirai vector, e.g., a UCvsgRNA lentivira! vector.
  • Each of the sgRNAs can target a different gene expressed in leukocytes, e.g. a gene involved in a leukocyte response or associated with an immune system disorder.
  • the sgRNAs can target one or more genes expressed in leukocytes so as to introduce loss-of-function mutatioii(s) and/or gain-of- function mutation(s).
  • the eukaryotic cell can be a mammalian leukocyte, e.g., a mouse leukocyte, such as a mouse leukocyte that is part of or derived from a transgenic mouse having cells that express Cas9.
  • the invention also comprehends a method for introducing one or more mutations ex vivo in Cas9-expressing leukocyte!
  • transgenic non-human mammal having leukocytes that express Cas9
  • the method can comprise delivering to leukocytes of or from the transgenic non-human mammal, and the transgenic non- human mammal is a transgenic mouse having leukocytes that express Cas9, e.g., a mouse that has had a Cas9 transgene knocked into the Rosa26 locus.
  • the Cas9 transgene can further comprise a Lox-Stop-poIyA-Lox(LSL) cassette thereby rendering Cas9 expression inducible by Cre recombinase.
  • the invention additionally comprehends a method for modeling an aberrant leukocyte response or a disease associated with leukocytes comprising introducing one or more, advantageously multiple, mutations ex vivo in Cas9 ⁇ expressing leukocyte(s), or in vivo in a transgenic non-human mammal having leukocytes that express Cas9, comprising delivering to the leukocyte(s) the vector as herein-discussed, wherein the specific mutations or precise sequence substitutions are or have been correlated to the disease.
  • This method can comprise delivering to leukocytes of the transgenic non-human mammal the vector, and the transgenic non-human mammal is a transgenic mouse having cells that express Cas9, e.g., a mouse that has had a Cas9 transgene knocked into the Rosa26 locus; and, the Cas9 transgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassette thereby rendering Cas9 expression inducible by Cre recombinase.
  • the invention also envisions an individualized or personalized treatment of a
  • 11 disease associated with leukocytes in a subject in need of such treatment comprising: (a) introducing one or more, e.g., multiple mutations ex vivo in Cas9 ⁇ expressing leukocyte(s), or in vivo in a transgenic non-human mammal having leukocytes that express Cas9, comprising delivering to leukocytes of the tissue, organ, cell or mammal the vector as herein-discussed, wherein the specific mutations or precise sequence substitutions are or have been correlated to the disease; (b) testing treatments) for the disease on the leukocytes to which the vector has been delivered that have the specific mutations or precise sequence substitutions correlated to the disease; and (c) treating the subject based on results from the testing of treatments) of step (b).
  • introducing one or more, e.g., multiple mutations ex vivo in Cas9 ⁇ expressing leukocyte(s), or in vivo in a transgenic non-human mammal having leukocytes that express Cas9 comprising
  • the method can comprise delivering to leukocytes of the transgenic non-human mammal the vector, and the transgenic non-human mammal is a transgenic mouse having leukocytes that express Cas9, and the disease is a leukocyte-associated disease, e.g., an immune disorder or cancer.
  • a leukocyte-associated disease e.g., an immune disorder or cancer.
  • the leukocyte can be a mammalian cell, e.g., a mouse ceil, such as a mouse cell that is part of a transgenic mouse having cells that contain or express or that are able to be induced to express or that conditionally express Cas9.
  • the invention also comprehends a method for introducing one or more, e.g., multiple mutations ex vivo in Cas9-expressing or -containing leukocytes or leukocytes containing or expressing or that are able to be induced to express or that conditionally express Cas9.
  • Such leukocytes can be transplanted into a non-human animal of the species from which they came.
  • a transgenic mouse having ceils that contain or express or that are able to be induced to express or that conditionally express Cas9 can have leukocytes removed therefrom.
  • Those cells can be transplanted into a second mouse that has had its immune system altered, e.g., a mouse that has been irradiated and/or had bone marrow removed and/or irradiated, whereby the second mouse has had its immune system altered.
  • That transplantatio can be before or after one or more, e.g., multiple, mutations are introduced via delivery of RNA(s), e.g., sgRNA(s) via vector(s) as herein discussed.
  • the second mouse may not be a transgenic mouse having ceils that contain or express or that are able to be induced to express or that conditionally express Cas9; but, it can be a mouse that has been altered such that its immune system has leukocytes that contain or express or that are able to be induced to express or that conditionally express Cas9, e.g., such leukocytes into which one or more, e.g., multiple, mutations are introduced.
  • the invention also comprehends a method for introducing one or more, e.g., multiple mutations, in vivo in a transgenic non-human animal, e.g., mammal (of the types herein discussed) having leukocyte(s) that express or contain Cas9 or that are able to be induced to express or that conditionally express Cas9, comprising delivering to leukocyte(s) of the tissue, organ, cell or mammal the vector as herein-discussed.
  • a transgenic non-human animal e.g., mammal (of the types herein discussed) having leukocyte(s) that express or contain Cas9 or that are able to be induced to express or that conditionally express Cas9
  • the transgenic non-human mammal can be a transgenic mouse having cells that express or contain Cas9 or that are able to be induced to express or that conditionally express Cas9, e.g., a mouse that has had a Cas9 transgene knocked into the Rosa26 locus.
  • the Cas9 transgene can further comprise a Lox- Stop-pofyA-Lox(LSL) cassette thereby rendering Cas9 expression inducible by Cre recombinase.
  • the invention additionally comprehends a method for modeling a leukocyte-associated disease, e.g., an immune disorder comprising introducing one or more, e.g., multiple, mutations ex vivo in a leukocytes that express or contain Cas9 or that are able to be induced to express or that conditionally express Cas9, or in vivo in a transgenic non-human animal, e.g., mammal (e.g., of the types herein discussed) having leukocytes that express or contain Cas9 or that are able to be induced to express or that conditionally express Cas9, comprising delivering to leukocytes of the animal, e.g., mammal a vector as herein-discussed, wherein the specific rautation(s) or precise sequence substitutions are or have been correlated to the leukocyte-associated disease, e.g., an immune disorder.
  • a transgenic non-human animal e.g., mammal (e.g., of the types
  • This method can comprise delivering to leukocytes of the transgenic non- human animal, e.g., mammal the vector, and the transgenic non-human animal, e.g., mammal is a transgenic mouse having leukocytes that express Cas9, e.g., a mouse that has had a Cas9 transgene knocked into the Rosa26 locus; and, the Cas9 transgene can further comprise a Lox- Stop ⁇ polyA-Lox(LSL) cassette thereby rendering Cas9 expression inducible by Cre recombinase.
  • the transgenic non-human animal e.g., mammal
  • the transgenic non-human animal e.g., mammal is a transgenic mouse having leukocytes that express Cas9, e.g., a mouse that has had a Cas9 transgene knocked into the Rosa26 locus
  • the Cas9 transgene can further comprise a Lox- Stop ⁇ polyA-L
  • the invention also envisions an individualized or personalized treatment of a leukocyte- associated disease, e.g., an immune disorder, in a subject in need of such treatment comprising: (a) introducing multiple mutations ex vivo in leukocytes that express or contain Cas9 or that are able to be induced to express or that conditionally express Cas9, or in vivo in a transgenic non- human animal, e.g., mammal having leukocytes that express or contain Cas9 or that are able to be induced to express or that conditionally express Cas9, comprising delivering to the leukocyte(s)a vector as herein-discussed, wherein the specific mutations or precise sequence substitutions are or have been correlated to the leukocyte-associated disease, e.g., an immune disorder or cancer; (b) testing treatment(s) for the leukocyte-associated disease, e.g., an immune disorder on the leukocytes to which the vector has been delivered that have the specific mutations or precise sequence substitutions
  • the method can comprise delivering to leukocytes of the transgenic non- human animal, e.g., mammal the vector, and the transgenic non-human animal, mammal can be a transgenic mouse having cells that express Cas9.
  • the invention comprehends a method of inducing one or more mutations in vitro or ex vivo in a eukaryotic cell as herein discussed comprising delivering to leukocyte(s) a vector as herein discussed.
  • the invention further comprehends a method of inducing one or more mutations in vivo in the non-human transgenic eukai ote, e.g., animal or mammal, as herein discussed comprising delivering to said non-human transgenic eukaiyote a vector as herein discussed.
  • the delivery can be to leukocytes.
  • the mutation(s) can include the introduction, deletion, or substitution of one or more nucleotides at each target sequence of cell(s) via the guide(s) or RNA(s) or sgRNA(s) resulting in loss-of-function and/or gain of function of the target(s).
  • the mutations can include the introduction, deletion, or substitution of 1-75 nucleotides at each target sequence of said cell(s) via the guide(s) or or RNA(s) sgRNA(s) resulting in loss- of-function or gain of function of the target(s).
  • the mutations can include the introduction, deletion, or substitution of 1, 5, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) or RNA(s) or sgRNA(s) resulting in loss-of-function or gain of function of the target(s).
  • the mutations can include the introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) or RNA(s) or sgRNA(s) resulting in loss-of- function or gain of function of the target(s).
  • the mutations include the introduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) or RNA(s) or sgRNA(s) resulting in loss-of-function or gain of function of the target(s).
  • the mutations can include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) or R A(s) or sgRNA(s) resulting in loss-of-function or gain of function of the target(s).
  • the mutations can include the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides at each target sequence of said cell(s) via the guide(s) or RNA(s) or sgR A(s) resulting in loss-of-function or gain of function of the target.
  • the method or mutation(s) thereof can induce a leukocyte-associated disease, e.g., an immune disorder or cancer.
  • the invention comprehends a method for identifying genes, mutations in genes or combinations thereof which are associated with or correlated to leukocyte-associated disease, e.g., an immune disorder or cancer, comprising a method of inducing one or more mutations in vitro or ex vivo in leukocyte(s) as herein discussed or inducing one or more mutations in vivo in a non-human transgenic eukaryote, e.g., animal or mammal, as herein discussed, and identifying leukocyte-associated disease, e.g., an immune disorder or cancer, resulting from such mutations(s).
  • the method can include control cell(s) or eukaryote(s) that do not receive introduction of said mutation(s).
  • the invention also comprehends a method for modeling leukocyte-associated disease, e.g., an immune disorder comprising inducing one or more mutations in vivo in a non-human transgenic eukaryote as herein discussed, e.g., deliverying to a non-human transgenic eukaryote one or more vectors or RNA(s) guide(s) or sgRNA(s) as herein discussed,
  • the invention additionally comprehends a method for generating non-human transgenic eukaryotes having leukocyte-associated disease, e.g., an immune disorder or cancer, by introducing, as herein discussed, the one or more mutations as herein discussed.
  • the invention additionally comprehends a non-human transgenic eukaryote having leukocyte- associated disease, e.g., an immune disorder or cancer; for instance, such a eukaryote produced by a method herein discussed.
  • a non-human eukaryote contains or expresses or conditionally expresses Cas9 and has a leukocyte-associated disease, e.g., an immune disorder.
  • the invention also comprehends a method for the identification of a treatment, e.g., chemical or gene thereapy treatment, for leukocyte-associated disease, e.g., an immune disorder or cancer, comprising applying, administering or delivering one or more treatments to the leukocyte-associated disease, e.g., an immune disorder or cancer, to non-human transgenic eukaryote as herein discussed and identifying whether the leukocyte-associated disease, e.g., an immune disorder or cancer, has improved.
  • a treatment e.g., chemical or gene thereapy treatment
  • leukocyte-associated disease e.g., an immune disorder or cancer
  • the method can comprise applying, administering or delivering different doses of the treatment and/or employing different routes of administration and/or different carriers or excipients and/or applying, administering or delivering at different time intervals, e.g., applying, administering or delivering different does at different time intervals.
  • the invention also comprehends a method of treatment of a leukocyte-associated disease, e.g., an immune disorder or cancer, e.g., such a leukocyte-associated disease, e.g., an immune disorder or cancer, identified in a herein method of identification or modeling, or another leukocyte-associated disease, e.g., an immune disorder or cancer, in a subject in need thereof, comprising treating the subject based on the results from the herein identification or modeling method.
  • a leukocyte-associated disease e.g., an immune disorder or cancer
  • kits e.g., comprising veetor(s) as herein discussed and one or more leukocyte(s) or non-human transgenic eukaryotes as herein discussed; and advantageously the kit includes instructions for performing a method as herein discussed.
  • the invention comprehends a method for modulating leukocyte activity, comprising delivering to a leukocyte a vector containing nucleic acid molecule(s), whereby the leukocyte contains or expresses or is able to be induced to express or conditionally expresses Cas9 and the vector expresses one or more R As to guide the Cas9 and optionally delivers donor template(s) to introduce mutations in one or more target genetic loci in the leukocyte, thereby modulating expression of one or more genes expressed in the leukocyte.
  • the RNAs to guide Cas9 can comprise chimeric single guide R A (sgRNA). Each sgRNA can be driven by a U6 promoter.
  • the vector can be a lentiv ral vector.
  • the vector can be a U6-sgRNA lentiviral vector.
  • the sgRNAs can target one or more genes expressed in the leukocyte so as to introduce loss-of- function mutations.
  • the leukocy e can be a mammalian leukocyte, e.g., a leukocyte derived from a non-human transgenic animal having cells that contain or express or are able to be induced to express or conditionally express Cas9, and the vector is delivered to the leukocyte ex vivo.
  • the non-human transgenic animal having cells that contain or express or are able to be induced to express or conditionally express Cas9 can be a mamma!
  • a mouse leukocyte such as a mouse leukocyte from a transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9.
  • the transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9 can comprises a mouse that has had a Cas9 transgene knocked into the Rosa26 locus.
  • the leukocyte can be a dendritic ceil.
  • the leukocyte can be a T cell.
  • the modulation can be an aberrant ieukocvte response or activation or inhibition or differentiation or exhaustion or adhesion or proliferation; or induction of rnutation(s) such as loss of function and/or gain of function mutations.
  • the invention includes genetic manipulation of cell line or primary cells or primary cells that were activated or differentiated to another cell type or cel l subset.
  • the invention also envisions a method of identifying a gene associated with a leukocyte response, comprising: (a) delivering to a plurality of leukocytes a plurality of different vectors containing nucleic acid molecuie(s), whereby the leukocytes contain or express or are able to be induced to express or conditionally express Cas9 and each vector expresses one or more RNAs (i) to guide the Cas9 and optionally delivers donor template(s) to introduce mutations in different target genetic loci in the leukocy es or (ii) to guide the Cas9 to activate or inhibit expression of a target gene, thereby modulating expression of different genes expressed in the leukocytes; (b) measuring a response in the leukocytes; and (c) isolating leukocyte(s) in which the response is modulated following delivery of the vector, thereby identifying gene(s) associated with the leukocyte response, in one embodiment, the genes associated with the leukocyte response are identified by
  • the leukocyte response can be activation, inhibition, differentiation migration, exhaustion, adhesion, or proliferation.
  • the leukocytes can comprise dendritic cells.
  • the response can comprise activation.
  • the dendritic cells can be stimulated with a cytokine, another ceil (e.g. activated T cell), agonist or antagonist antibodies targeting surface proteins, chemical agents, e.g.
  • a toxin such as diphtheria toxin or bacterial lipopolysacchari.de (LPS), or with any pathogen (virus, bacteria, fungus) or with other pathogen components (e.g., beta-glucan from fungi) or their synthetic mimics (e.g., poly I:C to mimic viral double stranded RNA).
  • the stimulation can be in the presence of an interleukin such as vnterleukin-10 (IL-! Q) or can be any combination of factors.
  • Activation can be measured by monitoring transcription factors, cytokines, cell surface markers or any molecule that is induced in the leukocyte cell, e.g. TNF-a or CD86 expression in the dendritic cells upon LPS stimulation.
  • the RNAs to guide Cas9 can comprise chimeric single guide RNA (sgRNA). Each sgRNA can be driven by a U6 promoter.
  • the vector can be a lentivirai vector, e.g., a U6-sgRNA lentiviral vector.
  • the sgRNAs can target one or more genes expressed in the leukocytes so as to introduce loss-of-function mutations. Gain of function mutations can also be introduced.
  • a decreased response in a leukocyte can be indicative that a gene mutated in the leukocyte by delivery of the vector is a positive regulator of the response.
  • the leukocytes can be mammalian leukocytes, e.g., mouse leukocytes.
  • the mouse leukocytes can be from a transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9.
  • the transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9 can comprises a mouse that has had a Cas9 transgene knocked into the Rosa26 locus.
  • the leukocytes can comprise T cells.
  • the response can comprise differentiation.
  • the T cells can be stimulated with an interleukin, e.g., interleukin-12.
  • the response can comprise differentiation to a Thl phenotype.
  • the T cells can be stimulated with i ter leukin-4.
  • the response can comprise differentiation to a Th2 phenotype.
  • the T cells can be stimulated with TGF- ⁇ .
  • the response can comprise differentiation to a Treg phenotype.
  • the T cells can be stimulated with TGF- ⁇ and an interleukin, e.g., interleukin-6.
  • the response can comprise differentiation to a Thl 7 phenotype.
  • the T cells can be from a transgenic mouse having cells that express Cas9 and an ovalbumin-specific ⁇ - ⁇ cell receptor.
  • a librar of vectors can be delivered to the leukocytes, each vector in the library expressing one or more RNAs targeted to a gene in a genome of the leukocytes, and wherein the library comprises at least one vector expressing an RNA targeted to each gene in the genome of the leukocytes.
  • the library comprises vectors expressing two, three, four, five or six or more RNAs targeted to each gene in the genome of the leukocytes.
  • the Cas9 is preferably a non-cutting enzyme, e.g. the Cas9 may be a mutant Cas9 lacking nuclease activity fused to a functional domain that activates or inhibits gene expression,
  • the invention further comprehends a method for modeling an aberrant leukocyte response or a disease associated with leukocytes, comprising delivering to leukocytes a vector containing nucleic acid rnolecule(s), whereby the leukocytes contain or express or are able to be induced to express or conditionally express Cas9 and the vector expresses one or more RNAs (i) to guide the Cas9 to introduce mutations in one or more target genetic loci in the leukocytes or (ii) to guide the Cas9 to activate or inhibit expression of a target gene, thereby modulating expression of one or more genes expressed in the leukocytes, and wherein expression of the one or more genes is associated with the response or disease.
  • the leukocytes can be derived from a non-human transgenic animal having cells that contain or express or are able to be induced to express or conditionally express Cas9, and the vector is delivered to the leukocyte ex vivo.
  • the transgenic non-human animal can be a transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9.
  • the transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9 can comprise a mouse that has had a Cas9 transgene knocked into the Rosa26 locus.
  • the aberrant leukocyte response can be deficient or excess activation, differentiation, inhibition, migration, exhaustion, adhesion, or proliferation.
  • the disease is an immune system disorder or cancer.
  • the Cas9 is preferably a non-cutting enzyme, e.g. the Cas9 may be a mutant Cas9 lacking nuclease activity fused to a functional domain that activates or inhibits gene expression.
  • the invention further envisions individualized or personalized treatment of a leukocyte-associated disease in a subject in need of such treatment comprising: (a) delivering to leukocytes ex vivo a vector containing nucleic acid molecule(s), whereby the leukocytes contain or express or are able to be induced to express or conditionally express Cas9 and the vector expresses one or more RNAs (i) to guide the Cas9 and optionally delivers one or more donor template(s) to introduce mutations in one or more target genetic loci or (ii) to guide the Cas9 to activate or inhibit expression of a target gene in the leukocytes, thereby modulating expression of one or more genes expressed in the leukocytes; (b) testing treatment(s) for the disease on the leukocytes to which the vector has been delivered ; and (c) treating the subject based on results from the testing of treatment(s) of step (b).
  • expression of one or more genes is correlated to the disease.
  • the leukocytes can be derived from a non-human transgenic animal having cells that contain or express or are able to be induced to express or conditionally express Cas9, and the vector is delivered to the leukocyte ex vivo.
  • the disease can be an immune system disorder or cancer.
  • the invention envisions an isolated leukocyte comprising a vector containing a nucleic acid moiecule(s), wherein the leukocyte contains or expresses or is able to be induced to express or conditionally express Cas9 and the vector encodes one or more RNAs capable of (i) guiding the Cas9 and optionally delivering one or more donor template(s) to introduce mutations in one or more target genetic loci or (ii) guiding the Cas9 to activate or inhibit expression of a target gene in the leukocyte, and thereby modulating expression of one or more genes expressed in the leukocyte.
  • the invention also envisions an isolated leukocyte comprising a vector containing a nucleic acid molecule(s), wherein the leukocyte contains or expresses or is able to be induced to express or conditionally express Cas9, (i) one or more target genetic loci in the leukocyte comprise mutations derived from Cas9 activity guided by one or more RNAs encoded by the vector or (ii) expression of one or more target genes in the leukocytes is activated by Cas9 guided by one or more RNAs encoded by the vector, and wherein expression of one or more genes expressed in the leukocyte is modulated.
  • the RNAs to guide Cas9 can comprise chimeric single guide RNA (sgRNA).
  • Each sgRNA can be driven by a U6 promoter.
  • the vector can be a lentiviral vector, e.g., a U6-sgRNA ientiviral vector.
  • the sgRNAs can target one or more genes expressed in the leukocyte so as to introduce loss-of-function mutations. Gain of function mutations can also be introduced.
  • the leukocyte can be a mammalian leukocyte.
  • the leukocyte can be derived from a non-human transgenic animal having cells that contain or express or are able to be induced to express or conditionally express Cas9.
  • the non-human transgenic animal having ceils that contain or express or are able to be induced to express or conditionally express Cas9 can be a mammal or a primate, or a rodent, or a mouse, or a rat or a rabbit, or a canine or dog, or a cow or bovine, or a sheep or ovine or a goat or a pig, or fowl or poultry, or a chicken, or a fish, e.g., a mouse leukocyte.
  • the mouse leukocyte can be from a transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9.
  • the transgenic mouse having cells that contain or express or are able to be induced to express or conditionally express Cas9 can comprise a mouse that has had a Cas9 transgene knocked into the Rosa26 locus.
  • the leukocyte can be a dendritic ceil.
  • the leukocyte can be a T cell.
  • the one or more genes can be associated with a leukocyte response or a leukocyte-associated disease.
  • the invention also envisions a non-human transgenic eukaryote or mammal having leukocytes that express or that are able to be induced to express or that conditionally express Cas9, and one or more mutation(s) in one or more gene(s) associated with a leukocyte response or a leukocyte-associated disease.
  • the invention also provides a method of monitoring an immune response in a subject comprising detecting a level of expression, activity and/or function of Olfr994, Olfr806, Olfr470, or Olfr394. or any one of the foregoing or any combination thereof.
  • the invention provides a method of detecting Olfr994, OIfr806, Olfr470, or 0(fr394 in any combination of thereof at a first time point, or detecting a level of expression, activity and/or function of one or more signature genes or one or more products of Olfr994, Olfr806, Olfr470, or Olfr394 or any one of the foregoing or any combination thereof and detecting Olfr994, Olfr806, Oifr470, or 01 r394 Oifr394 or any one of the foregoing or any combination thereof at a second time point, and comparing the first detected level of expression, activity and/or function with the second detected level of expression, activity and/or function, wherein a change in the first and second detected levels indicates a change in the immune response in the subject.
  • the invention provides for modulating an immune response by- treatment with iigands for olfactory receptors.
  • the olfactory receptors may be Olfi-994, Qlfr806, OIfr470, or OIfr394,
  • An immune response may be activated or repressed by using Iigands specific for positive or negative receptors, in one embodiment, the immune response is modulated by smell. Not being bound by a theory, different smells will activate d fferent olfactory receptors and will result in different immune responses.
  • Figure 1 shows a schematic of experimental flow in ex vivo genome editing of primary immune cells derived from constitutive Cas9-expressing mice.
  • Figure 2 shows a flow cytometr histogram of bone marrow cells from constitutive Cas9-expressing (green) and wild-type (blue) mice, showing Cas9-P2A-EGFP expression only in Cas9 mice. Data are plotted as a percentage of the total number of cells.
  • Figure 3 shows sgRNA design for targeting the mouse Myd88 locus.
  • Figure 4 shows sgRNA design for targeting the mouse A20 locus.
  • Figure 5 shows Myd88 indel analysis of constitutive Cas9-expressing DCs transduced with either a _ SS ⁇ targeting sgRNA (sgMyd88-l and sgMyd88-2) or controls (CTR, average of four control sgRNAs), showing indel formation only in A ⁇ 3 ⁇ 43 ⁇ 4# ⁇ targeted cells. Data are plotted as the percent of Illumina sequencing reads containing indels at the target site. Mutations are categorized as frame-shift (fs, yellow bar) or non-frame-shift (nfs, orange bar).
  • Figure 6 shows A20 indel analysis of constitutive Cas9 -expressing DCs transduced with either an ..4 0-targeting sgRNA (sgA20-l) or controls (CTR, average of four control sgRNAs), showing indel formation only in ,420-targeted cells. Data are plotted as the percent of Illumina sequencing reads containing indels at the target site. Mutations are categorized as frame-shift (fs, yellow bar) or non-frame-shift (nfs, orange bar).
  • FIG. 7 shows Myd88 mRNA quantification of constitutive Cas9-expressing DCs transduced with either jYiSS-targeting sgRNA (sgMyd88-l or sgMyd88-2) or controls (CTR, average of six control sgRNAs), showing reduced expression only in Myd88-targetGd cells. Data are plotted as Myd88 mRNA levels from Nanostring nCounter analysis.
  • Figure 8 shows an immunoblot of constitutive Cas9-expressing DCs transduced with either Myd88-targeting sgRNA (sgMyd88 ⁇ l or sgMyd88-2) or controls (four control sgRNAs), showing depletion of Myd88 protein only in Myd88-targeted cells.
  • Beta-actin was used as a loading control. * overexposed, repeated measurement.
  • Figure 9 shows nanostring nCounter analysis of constitutive Cas9-expressing DCs transduced with either ji SS-targeting sgRNA (sgMyd88-l or sgMyd88-2) or shRNA (shMyd88), or / 20-targeting sgRNA (sgA20-l or sgA20-2) or shR A (shA20), showing an altered LPS response.
  • Inset the cluster showing the highest difference between Myd88- and A20- targeting sgRNAs, including key inflammatory genes (ILla, ILlb, Cxcll, Tnf, etc.). Red: high; blue: low; white: unchanged; based on fold change relative to measurements with six control
  • Figure 10 shows flow cytometry scatter plots and histograms showing Cas9-P2A- EGFP positive immune cells of various immune cell types from constitutive Cas9-expressing but not wild-type (WT) mice.
  • Figure 11 shows a schematic representation of a genome-wide CRISPR screen in primary mouse dendritic cells.
  • Figure 12 shows flow cytometry and bin selection in three CRISPR screens done in mouse primary dendritic cells. After 9 days of differentiation and expansion, the cells were 1) Stimulated with LPS for 8 hours and stained for TNF-a. 2) Stimulated with LPS for 24 hours and stained for CD86. 3) Stimulated with LPS and IL-10 for 24 hours and stained for CD-86.
  • Figure 13 shows the levels of guides in the input library of vectors or of guides that were sequenced from ceils after 9 days of expression.
  • Figure 14 shows the fold changes of guide levels in experiment 1 (y axis) as per Figure 12 as a function of the mean (x axis).
  • the fold change in guide level is between the high and the low bins of TN F a expression.
  • Known regulators of the LPS response are highlighted.
  • Figure 15 shows the density plot of the fold changes in guide levels. Blue: fold changes between high and low TNF-a bins; pink: fold changes in two repeats of the control experiment.
  • Figure 16 depicts the overlap between the top hits of two biological repeats of experiment 1), day 9 is presented as a control.
  • Figure 17 shows a schematic representation of a genome -wide CRISPR screen in T cells.
  • FIG. 18A-E A Genome wide pooled CRISPR screen in mouse primary DC.
  • A Single gene CRISPR knockout of known regulators of the LPS response in primary DCs affects Tnf expression. Shown are flow cytometry measurements of intracellular Tnf following 8h of LPS stimulation in BMDCs with sgRNAs targeting the positive regulators Tlr4 (black) and Myd88 (blue), the negative regulator Zfp36 (red) and a non-targeting control sgRNA (grey).
  • B Experimental design for a genome wide CRISPR screen to identify regulators of the LPS response in primary mouse BMDCs.
  • C sgRNAs targeting essential genes are significantly underrepresented in the 'Pre-LPS' versus "Input' sample. Applicants averaged the sgRNA Z-scores of the ratio Input/Pre-LPS for the top four sgRNAs that target the same gene (Experimental Procedures). Shown are cumulative distribution function (CDF) plots of the gene level Z-score distribution of 'all genes' detected (grey), 'all essential' (purple) and 'core essential' (black) genes as defined in (Hart et al., 2014), and all genes associated with the Gene Ontology annotation 'Translation' (blue).
  • CDF cumulative distribution function
  • (D) sgRNAs targeting known positive and negative regulators are significantly over- and underrepresented, respective!)', in 'Tnf 10 ' versus 'Tnf* 1 " bins.
  • Negative regulators were tested with saturating (100%) and - in some cases (numbers in parenthesis) with low (20%) - LPS concentrations.
  • B Most components of the core TLR pathway are identified by the screen and validated by the individual assay. Left: Shown are all components of the TLR pathway as defined by EGG that directly link LPS and Tnf, and their rank in the genome wade CRISPR screen (blue scale).
  • Figure 20A-F The validated positive regulators partition into key modules based on their effect on four protein markers and RN.A expression profiles.
  • A Positive regulators are grouped by distinct effects on protein expression. For each positive regulator (rows) shown are its effects (blue: significantly reduces; red: significantly increases; Experimental Procedures) on the expression of each of five proteins (columns) measured by staining with antibodies (Experimental Procedures). Based on this matrix, three broad categories of responses can be defined, each preferential!' associated with distinct proteins (color bar on left: turquoise: known LPS response genes; purple: members of the OSTc; petrol blue: members of the PAFc).
  • B Effects of key regulator groups on protein markers.
  • each marker (X axis), shown are two Violin plots of the distribution of Z scores of true positive regulators of Tnf (left) or of non- targeting control sgR As (right). Key functional groups are colored as in (A).
  • D-F Positive regulators partition to modules based on their effect on niRNA profiles over time.
  • FIG. 21A-E The OST complex strongly affects the BMDC inflammatory response.
  • A Positive regulators in the context of the secretory pathway. Left: Shown are proteins (blue rectangles) from the OST complex and the secretory pathway that ranked high in the genome wide CRISPR screen. Right: Flow cytometry staining of intracellular Tnf for each targeted gene (blue) compared to sgRNA controls (black).
  • B - E Specific impact of OSTc perturbation on inflammatory gene expression at (B) basal state (s Oh ): (C) 2 hours post LPS; (D) 4 hours post LPS.
  • the genes are shown in the same order in all panels, and marked on left for their membership in the anti-viral module (orange), peaked inflammatory module and sustained inflammatory module (green) and ER-stress response module (green).
  • FIG. 22A-J The Paf complex strongly affects the LPS response.
  • A,B PAFc component knockout reduced Tnf expression. Shown are flow cytometr stainings of intracellular Tnf from cells with sgRNAs targeting Pafl (A) or Rtfl (B) (blue), compared to sg NA controls (black).
  • C-F Knockout of PAFc members affects mainly the antiviral response, while knockout of OSTc members mainly affects the inflammatory response.
  • a score of 0 reflects a similar response as in non-targeting controls. Positive and negative values indicate an increased and reduced response, respectively.
  • G, H Immunopurification (IP) followed by LC-MS/MS to identify Pafl or Rtfl interactors. Scatter plot of two independent IPs for Pafl (G) or Rtfl (H). Shown are the Iog2 ratios of the Pafl or Rtfl IP relative to a control IP. Blue dots indicate interactors that have been tested by individual sgRNA experiments for an effect on Tnf expression, with the IP target (Pafl or Rtfl) marked in bold. Blue dots with black borders in G indicate previously known PAFc members.
  • FIG. 23 A- H Quality measures of a Genome wide pooled CRISPR screen in mouse primary DC.
  • A Reproducibility. Shown are scatterpiots comparing the log 2 (quantile normalized read counts) of sgRNAs between two replicate screens for the lowest bin (left), 2 na lowest bin (middle), and top bin (right). Pearson con-elation coefficient (r) is shown in top left corner.
  • B-E Top ranked screen hits compare well between the DESeq and Z-score approaches.
  • the Spearman rank correlation coefficient (p) is noted.
  • sgRNAs that target translation genes are enriched in the "Input” library versus "Pre-LPS".
  • Left Scatterplot compares the normalized fold change in sgRNAs (Input / Pre-LPS) to the mean abundance in the two libraries.
  • Middle and Right Distribution of the normalized fold change (Input Pre-LPS; Y axis) in either sgRNAs (middle) or genes (right: mean of the top 4 ranked sgRNAs).
  • Orange translation genes; black: ail genes; grey: non- targeting controls.
  • G sgRNAs targeting known regulators of LPS response are highly significant in DE-Seq analysis.
  • MA-piots compare for either sgRNAs (left) or genes (right), the DE-Seq calculated fold-change between TNF l and TNF low (Y axis) to the mean abundance of the sgRNA or gene.
  • Screen hits are more likely to be expressed post-LPS and at higher level than al l other genes.
  • Violin plots show the distribution of mean expression (Y axis) along LPS stimulation (Oh, 2h, 4h, 6h) in control ceils, for top- 169 hits (right) and for all other genes (left).
  • FIG. 24A-D Analysis of secondary screen,
  • A-B sgRNAs targeting known regulators of LPS response (orange) have highly significant Z-scores, compared to other genes (black) and non-targeting controls (grey). Shown are MA-plots that relate for either sgRNAs (A) or genes (B) the z-score calculated fold-change between TNF" 1 and TNF low (Y axis) to the mean abundance of the sgRNA or gene (X axis).
  • C Top ranked screen hits compare well between the DE and ZS approaches.
  • FIG. 25A-E Partitioning of the validated positive regulators into key modules by their effect on protein markers and RNA profiles,
  • A Positive regulators group by distinct effects on protein expression.
  • For each sgRNA targeting a positive regulator (rows) shown are its effects (Z score for each marker compared to non-target sgRNA; Experimental Procedures) on the expression of each of five proteins (columns) measured by staining with antibodies (Experimental Procedures). Three broad categories of responses can be defined, each preferentially associated with distinct proteins.
  • sgRNAs were collapsed to score gene level effects as in Figure 3A,
  • B-E Positive regulators partition to modules based on their effect on mRNA profiles over time.
  • FIG. 26 Knockout of members of the OSTc module reduces the expression of five key protein markers.
  • Each panel shows flow cytometry staining of the levels (X axis) of each of five protein markers (from left to right: Tnf, Cdl lc, Cdl4, Mipl , 116) in ceils with individual sgRNA targeting specific genes (colored histogram; gene name in top left corner) compared to sgRNA controls (black curves).
  • Data is shown (from top to bottom) for three representative members of OSTc (Ddost, Rpnl, Rpn2), and two other members of the module: Alg2 and Tmem258.
  • FIG. 27A-E The PAFc module.
  • A-D Each panel shows flow cytometr staining of either intracellular Tnf levels (A, B, D; X axis) or Cdl lc levels (C, X axis) for each targeted gene (colored histogram; gene name in top left corner) compared to sgRNA controls (black curves).
  • E Validation of Pafl and Auh interaction by Western blot. Shown are the immunopurifications (IPs) in BMDCs performed with either Pafl antibody (PAF) or IgG antibody (Control). Input or IP samples were incubated with either Pafl antibody (top) or Auh antibody (bottom). IPs were performed in unstimulated BMDCs (LPS "-") or in BMDCs stimulated with LPS for 2h (LPS "+ ").
  • FIG. 28A-E Assessing false negatives and true positives in individual validation experiments. Each panel shows flow cytometry staining of intracellular Tnf levels (X axis) for each targeted gene (colored histogram; gene name in top left comer) compared to sgRNA controls (black curves).
  • A, B Determining the false negative rate. Known regulators of the LPS response that did not rank within the top- 100 in the screen were tested individually by single sgRNAs followed by Tnf staining and flow cytometry (see Figure 2A).
  • C-E Sensitive validation of novel negative regulators requires screening at unsaturated levels of Tnf.
  • E BMDC transduced with an sgRN A targeting Stat5b were stained with Cdl l c antibody.
  • Figure 29 A protein interaction map associating known regulators and validated all the validated positive and negative regulators from the screen. Applicants attempted to directly connect 75 Tnf regulators (57 positive and 18 negative) found in this study (beige nodes) to 35 known members of LPS-TLR4 pathway (boldfaced labels) and 23 relevant transcription factors (red nodes and Cebpb, Rela, and Nfkbl) previously identified (see Experimental procedure). Applicants used information from protein-protein interaction databases and AP-MS data collected in this study. The members of modules identified in various screens (see Fig.
  • node rim colors are marked by node rim colors as follows: PAF complex known members (dark blue) and other module members (light blue); OST complex (dark and light purple); LPS-TLR4 module members (dark green).
  • Node sizes are linearly proportional to the absolute values of scores in the primary TNF screen.
  • Rounded square nodes indicate transcription factors. Red edges indicate interactions found in the AP-MS experiment (bright red - novel, dark red - confirming).
  • FIG. 30A-C Mutation analysis of sequencing sorted mutants.
  • BMDCs were transduced with sgRNA. targeting the indicated gene ((A) Pafl , (B) Dadl and (C) Cdl4; marked on top), stimulated with LPS, and flow-sorted based on high or low Tnf antibody staining.
  • Genomic DNA was isolated from sorted cells ("low Tnf and "high Tnf), unsorted cells (“Presort”), and cells without relevant sgRNA (control; only in A and C). The region surrounding the sgRNA target site was amplified and sequenced to analyze mutational composition of the targeted locus.
  • FIG. 31 Effect of Brefeldin on the expression of the different modules. Violin plots show for each validated regulator (dot) the ratio of expression values (log(TPM+)) when comparing between Brefeldin vs. no Brefeldin conditions, in each of 3 modules and TNF (X axis).
  • Applicants adapt the CRISPR-Cas9 technology to develop a marker based genome-wide knockout screen in primary mammalian cells derived from Cas9-expressing transgenic mice, and applied it to dissect gene regulatory circuits in the response of DCs to LPS.
  • Applicants infect DCs with a pooled, genome-wide library of ientiviruses, stimulate them with LPS, and monitor their responses by intra-ceilular staining for the inflammatory cytokine TNF, a major marker of the early response to LPS.
  • Applicants use Fluorescence Activated Cell Sorting (FACS) to isolate cells that failed to respond to LPS (i.e., caused by a loss-of-function mutation in a positive regulator) or that responded more strongly (i.e., caused by a loss-of-function mutation in a negative regul ator), and determine their resident sgRNAs by deep sequencing. Many of the key known regulators of TLR signaling are recovered as the highest regulators in the screen.
  • Applicants analyze each of 57 validated positive regulators by their effect on four additional protein markers reflecting different aspects of DC biology, and on genome wide expression profiles at four time points post LPS, identify 3 key functional modules, each with a distinct regulatory effect.
  • modules allow one to relate new regulators to their functions by a guiIt-by-/iiR6 /o/ia -association approach, as well as highlight novel distinctive functions in the response. These include roles for the members of the PAF complex, involved in RNA transcription and processing, and of the OST complex important for protein glycosylation. Applicants identify new facets in the complex response of immune cells to pathogens, and provide a general strategy for systematically dissecting circuits in other primary mammalian cells.
  • Applicants provide a new method to investigate modules and factors in immune pathways applicable across diverse biological systems.
  • the methods provided are coupled with advances in single cell profiling that bridge the gap between genome -wide pooled screens and deep molecular readouts.
  • methods such as DropSeq may be employed, Drop-seq is a method which enables highly parallel analysis of individual cells by RNA-seq. Specifically, the RNA content of tens and hundreds of thousands of individual human cells are profiled quickly and inexpensively.
  • Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 108 discrete biological entities (including, but not limited to, individual cells or organelles) to be screened in a single day. Compartmentalization in droplets increases assay sensitivity by increasing the effective concentration of rare species and decreasing the time required to reach detection thresholds. Droplet microfluidics combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-ceil and single-molecule assays. (Klein et al,, 2015, Cell 161 , 1187-1201),
  • Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F., Nature. Jan 29;517(7536):583-8 (2015).
  • Jiang et al. used the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coii.
  • CRISPR clustered, regularly interspaced, short palindromic repeats
  • the approach relied on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems.
  • the study reported reprogramming dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates.
  • Nishimasu et al. reported the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A° resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNAiDNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and D ' NA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and non-complementary strands of the target D ' NA, respectively.
  • the nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • Doench et al. created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry.
  • the authors showed that optimization of the PAM improved activity and also provided an on-line tool for designing sgRNAs.
  • onermann et al. (2015) discusses the ability to attach multiple effector domains, e.g., transcriptional activator, functional and epigenomic regulators at appropriate positions on the guide such as stem or tetraloop with and without linkers.
  • effector domains e.g., transcriptional activator, functional and epigenomic regulators
  • Chen et al relates to multiplex screening by demonstrating that a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes regulating lung metastasis.
  • the CRISPR-Cas or CRISPR system is as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667) and refers collectively to transcripts and other elements involved in the expression of or directing the activity of CR ISPR- associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
  • RNA(s) as that term is herein used (e.g., RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DN A or RN A polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • direct repeats may be identified in silica by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%», 95%», 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), OustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Iliumiiia, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), OustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Iliumiiia, San
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex may be provided to a host cell having the corresponding target sequence, such as by trans fection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • a guide sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome.
  • the target sequence is comprised in a gene expressed in leukocytes, particularly a gene associated with leukocyte responses or a leukocyte-associated disease.
  • a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the guide sequence participate in self-complementary base pairing when optimally folded.
  • Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. Gruber et aL ' , 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1 ) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence.
  • degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as seff-complementarity within either the tracr sequence or tracr mate sequence.
  • the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%», 60%, 70%, 80%, 90%», 95%, 97.5%, 99%», or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the transcript or transcribed polynucleotide sequence has at least two or more hairpins.
  • the transcript has two, three, four or five hairpins.
  • the transcript has at most five hairpins.
  • the portion of the sequence 5' of the final "N" and upstream of the loop corresponds to the tracr mate sequence
  • the portion of the sequence 3' of the loop corresponds to the tracr sequence
  • N represents a base of a guide sequence
  • the first block of lower case letters represent the tracr mate sequence
  • the second block of lower case letters represent the tracr sequence
  • the final poly-T sequence represents the transcription terminator: (1) N N N N N N N N N N NNNNNgtttttgiactc caagatttaGAi _i taaatcttgcagaagciacaaagataaa ggcttcatgccgaaatcaacaccctgtcatttt ggcagggtgtttcg ⁇ ttta
  • sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPRl.
  • sequences (4) to (6) are used in combination with Cas9 from S, pyogenes.
  • the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence,
  • candidate tracrRNA may be subsequently predicted by sequences that fulfill any or all of the following criteria: 1. sequence homology to direct repeats (motif search in Geneious with up to 18-bp mismatches); 2. presence of a predicted Rho- independent transcriptional terminator in direction of transcription; and 3. stable hairpin secondary structure between tracrRNA and direct repeat.
  • 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.
  • all 3 criteria may be used.
  • chimeric synthetic guide RNAs may incorporate at least 12 bp of duplex structure between the direct repeat and tracrRNA.
  • CRISPR enzyme mRN A and guide RNA delivered For minimization of toxicity and off-target effect, it will be important to control the concentration of CRISPR enzyme mRN A and guide RNA delivered.
  • Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci.
  • deep sequencing can be used to assess the level of modification at the following two off-target loci, 1 : 5 '-GAGTCCTAGCAGGAGAAGAA-3 ' and 2: 5 '-GAGTCTAAGCAGAAGAAGAA- 3'.
  • concentration that gives the highest level of cm-target modification while minimizing the level of off-target modification should be chosen for in vivo delivery.
  • CRISPR. enzyme nickase mRNA for example S.
  • pyogenes Cas9 with the D10A mutation can be delivered with a pair of guide RNAs targeting a site of interest.
  • the two guide RNAs need to be spaced as follows.
  • Guide sequences and strategies to mimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667).
  • the CRISPR system is derived advantageously from a type II CRISPR system.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage.
  • Cas proteins include Casl , Casl B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, CsxT7, Csxl 4, CsxlO, Cs l6, CsaX, Csx3, Csxi, Csxl5, Csfl, Cs£2, CsG, Csf4, homoiogues thereof, or modified versions thereof.
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the CRISPR enzyme directs cleavage of one or both strands within about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • two or more catalytic domains of Cas9 may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity.
  • a DIOA mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.
  • a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non- mutated form of the enzyme; an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form.
  • the enzyme is not SpCas9
  • mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools).
  • any or all of the following mutations are preferred in SpCas9: DIOA, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged.
  • the same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also preferred.
  • Particularly preferred are D10 and H840 in SpCas9.
  • residues corresponding to SpCas9 DI G and H840 are also preferred.
  • Orthologs of SpCas9 can be used in the practice of the invention.
  • a Cas enzyme may be identified Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system.
  • the Cas9 enzyme is from, or is derived from, spCas9 (S. pyogenes Cas9) or saCas9 (S, aureus Cas9).
  • StCas9 refers to wild type Cas9 from S. thermophiius, the protein sequence of which is given in the SwissProt database under accession number G3ECR1.
  • S. pyogenes Cas9 or spCas9 is included in SwissProt under accession number Q99ZW2.
  • Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent.
  • residue nurnberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.
  • this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, StlCas9 and so forth.
  • Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 generates double stranded breaks at target site sequences which hybridize to 20 nucleotides of the guide sequence and that have a protospacer-adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be determined as described herein) following the 20 nucleotides of the target sequence.
  • PAM protospacer-adjacent motif
  • the CRISPR system small RNA- guided defence in bacteria and archaea, Mole Cell 2010, January 15; 37( 1): 7.
  • the type II CRISPR locus from Streptococcus pyogenes SF370 which contains a cluster of four genes Cas9, Casl, Cas2, and Csnl , as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30bp each).
  • DSB targeted DNA double-strand break
  • tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crR A and the protospacer DNA.
  • Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer.
  • Cas9 may be constitutiveiy present or inducibly present or conditionally present or administered or delivered. Cas9 optimization may be used to enhance function or to develop new functions, one can generate chimeric Cas9 proteins. And Cas9 may be used as a generic DNA binding protein.
  • a CRISPR complex comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins
  • formation of a CRISPR complex results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild- type tracr sequence (e.g.
  • a wild-type tracr sequence may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence,
  • a codon optimized sequence is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known.
  • an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.
  • processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes may be excluded.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g.
  • Codon bias differences in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RN A
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the "Codon Usage Database” available at www.kazusa.orjp/codon,'' (visited Jul. 9, 2002), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons correspond to the most frequently used codon for a particular amino acid.
  • a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. zero or at least one or more NLS at the amino-temiinus and zero or at one or more NLS at the carboxy terminus).
  • the CRISPR enzyme comprises at most 6 NLSs, In some embodiments, an NLS is considered near the - or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence RPAAT AGQAK K ); the c-myc N LS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence
  • the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.
  • strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors.
  • Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to the CR ISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI).
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DN A cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.
  • an assay for the effect of CRISPR complex formation e.g. assay for DN A cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity
  • aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5 ' overhangs to reanneal and ligate or the activity or function of the gene product being al tered or the expression of the gene product being increased.
  • the gene product is a protein. Only sgRNA pairs creating 5 ' overhangs with less than 8bp overlap between the guide sequences (offset greater than -8 bp) were able to mediate detectable indel formation.
  • each guide used in these assays is able to efficiently induce indels when paired with wi ldtype Cas9, indicating that the relative positions of the guide pairs are the most important parameters in predicting double nicking activity.
  • Cas9n and Cas9H840A nick opposite strands of DNA
  • substitution of Cas9n with Cas9 840A with a given sgRNA pair should have resulted in the inversion of the overhang type; but no indel formation is observed as with Cas9B840A indicating that Cas9H840A is a CRISPR enzyme substantially lacking all DNA cleavage activity (which is when the DN A cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non- mutated form of the enzyme; whereby an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mut
  • a recombination template is also provided.
  • a recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide.
  • a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex.
  • a template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length.
  • the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence.
  • a template polynucleotide When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about I, 5, 10, 15, 20, or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operabiy linked to separate regulatory elements on separate vectors.
  • RNA(s) of the CRISPR. System can be delivered to a transgenic Cas9 animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses Cas9.
  • two or more of the elements expressed from the same or different regulatory elements may be combmed in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector, CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream” of) or 3' with respect to ("downstream" of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or ail in a single intron) ,
  • the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
  • a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • insertion sites such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • one or more insertion sites e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell.
  • a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site, in such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these.
  • a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
  • a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.
  • a vector comprises a regulatory element operably linked to an enzyme- coding sequence encoding a CRISPR enzyme, such as a Cas protein.
  • CRISPR enzyme or CRISPR enzyme mRNA or CRISPR guide RNA or RNA(s) can be delivered separately; and advantageously at least one of these is delivered via a nanoparticle complex.
  • CRISPR enzyme mRNA can be delivered prior to the guide RNA to give time for CRISPR. enzyme to be expressed.
  • CRISPR enzyme mRNA might be administered 1-12 hours (preferably around 2-6 hours) prior to the administration of guide RNA.
  • CRISPR enzyme mRNA and guide RNA can be administered together.
  • a second booster dose of guide RNA can be administered 1-12 hours (preferably around 2-6 hours) after the initial administration, of CRISPR enzyme mRNA + guide RNA. Additionai administrations of CRISPR enzyme mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome modification.
  • the invention provides methods for using one or more elements of a CRISPR system.
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide.
  • the CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types.
  • modifying e.g., deleting, inserting, translocating, inactivating, activating
  • the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene therapy, gene screening, dmg screening, disease diagnosis, and prognosis.
  • An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide.
  • the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
  • this invention provides a method of cleaving a target polynucleotide. The method comprises modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and effect cleavage of said target polynucleotide.
  • the CRISPR complex of the invention when introduced into a cell, creates a break (e.g., a single or a double strand break) in the genome sequence.
  • a break e.g., a single or a double strand break
  • the method can be used to cleave a disease gene in a cell.
  • the break created by the CRISPR complex can be repaired by a repair processes such as the error prone non-homologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR).
  • NHEJ error prone non-homologous end joining
  • HDR high fidelity homology-directed repair
  • the HDR process is used to modify genome sequence.
  • an exogenous polynucleotide template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell.
  • the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome.
  • a donor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PGR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • the exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene).
  • the sequence for integration may be a sequence endogenous or exogenous to the cell.
  • Examples of a sequence to be integrated include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA).
  • the sequence for integration may be operably linked to an appropriate control or regulatory sequence or sequences.
  • the sequence to be integrated may provide a regulatory function.
  • the upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide.
  • the upstream sequence is a nucleic acid sequence that shares sequence similarity with the genome sequence upstream of the targeted site for integration.
  • the downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration.
  • the upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the targeted genome sequence.
  • An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
  • the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp.
  • the exogenous polynucleotide template may further comprise a marker.
  • a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers.
  • the exogenous polynucleotide template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et ai, 2001 and Ausubel et al., 1996), In a method for modifying a target polynucleotide by integrating an exogenous polynucleotide template, a double stranded break is introduced into the genome sequence by the CRISPR complex, the break is repaired via homologous recombination of an exogenous polynucleotide template such that the template is integrated into the genome.
  • this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises increasing or decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide.
  • a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does.
  • a protein or microRNA coding sequence may be inactivated such that the protein or microRNA or pre-microRNA transcript is not produced.
  • a control sequence can be inactivated such that it no longer functions as a control sequence.
  • control sequence refers to any nucleic acid sequence that effects the transcription, translation, or accessibility of a nucleic acid sequence. Examples of a control sequence include, a promoter, a transcription terminator, and an enhancer.
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protem) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • a “disease-associated" gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown, and may be at a normal or abnormal level .
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex.
  • PAM protospacer adjacent motif
  • the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Examples of PAM sequences are given in the examples section below, and the skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • Similar considerations and conditions apply as above for methods of modifying a target polynucleotide. In fact, these sampling, culturing and re -introduction options apply across the aspects of the present invention.
  • the invention provides for methods of modifying a target polynucleotide in a leukocyte, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a leukocyte or population of leukocytes from a human or non-human animal, and modifying the leukocytes. Culturing may occur at any stage ex vivo. The leukocytes may even be re-introduced into the non-human animal or mammal.
  • the CRISPR complex may comprise a CRiSPR enzyme complexed with a guide sequence hybridized to a target sequence, wherein said guide sequence may be linked to a tracr mate sequence which in turn may hybridize to a tracr sequence.
  • Transgenic non-human eukaryotic organisms e.g., animals are also provided in an aspect of practice of the instant invention.
  • Preferred examples include animals comprising Cas9, in terms of polynucleotides encoding Cas9 or the protein itself.
  • the invention involves a constitutive or conditional or inducible Cas9 non-human eukaryotic organism, such as an animal, e.g., a primate, rodent, e.g., mouse, rat and rabbit, are preferred; and can include a canine or dog, livestock (cow / bovine, sheep / ovine, goat or pig), fish, fowl or poultry, e.g., chicken with it mentioned that it is advantageous if the animal is a model as to a human or animal genetic disease or condition, such as a disease associated with leukocytes, as use of the non-human eukaryotic organisms in genetic disease or condition modeling, e.g., via inducing a plurality, e.g., 1 or more, e.g., a plurality or multiple, such as 3 or more, e.g., 3 to 50 mutations correlated or associated with a genetic disease or condition, such as a disease associated with leukocytes, are preferred.
  • transgenic mice with the constructs as exemplified herein one may inject pure, linear DNA into the pronucleus of a zygote from a pseudo pregnant female, e.g. a CB56 female. Founders may then be identified, gertotyped, and backcrossed to CBS 7 mice. The constructs may then be cloned and optionally verified, for instance by Sanger sequencing. Knock ins are envisaged (alone or in combination).
  • Example 1 provides a knockin Cas9 mouse and to generate a Cas9 knock in mice one may target the same constitutive and conditional constructs to the Rosa26 locus, reference is made to WO 2014/093622 (PCT/US 13/74667), incorporated herein by reference.
  • the Cas9 conditional expression construct can be activated by co-expression with Cre. Correctly targeted Rl mESCs can have active Cas9 when Cre is expressed. Because Cas9 is followed by the P2A peptide cleavage sequence and then EGFP Applicants identify successful expression by observing EGFP. Applicants have shown Cas9 activation in mESCs.
  • the conditional Cas9 mouse can be crossed with a mouse that ubiquitously expresses Cre (ACTB-Cre line) and may arrive at a mouse that expresses Cas9 in ever ⁇ ' cell. The deliver ⁇ ' of RNA(s) to guide Cas9, e.g.
  • CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA) can induce genome editing in embryonic or adult mice.
  • sgRNA single guide RNA
  • the conditional Cas9 mouse is crossed with a mouse expressing Cre under a tissue specific promoter, there should only be Cas9 in the tissues that also express Cre. This approach may be used to edit the genome in only precise tissues (e.g. in leukocytes) by delivering chimeric RNA to the same tissue.
  • the conditional Cas9 mouse is broadly applicable for many areas of biology and its uses as described herein provides many utilities.
  • Cas9 provides an effective way to model multiple genetic changes
  • Cas9 delivery has challenges, including limitations due to the significant size of Cas9, and possible extra biosafety practices.
  • the Cas9 transgenic eukaryote e.g., mouse provides a most effective method for modeling of aberrant leukocyte responses, leukocyte-associated diseases and other areas of tissue-specific biological studies.
  • Applicants' approach provides a substantial packaging capacity for both sgRNAs as well as reporters and other modulators, etc.
  • These features include efficient editing of cellular subtypes (e.g.
  • the invention involves a non-human eukaryote, animal, mammal, primate, rodent, etc or leukocyte thereof that may be used as a disease model or a model of aberrant leukocyte responses.
  • disease refers to a disease, disorder, or indication in a subject.
  • a method of the invention may be used to create a non- human eukaryote, e.g., an animal, mammal, primate, rodent or leukocyte that comprises a modification, e.g., 3-50 or more modifications, in one or more nucleic acid sequences associated or correlated with a leukocyte response or disease, e.g., a leukocyte associated disease.
  • a mutated nucleic acid sequence may be associated or correlated with a disease or expressed in leukocytes and may encode a disease associated protein sequence or may be a disease associated or correlated control sequence.
  • the leukocyte may be in vivo or ex vivo.
  • a cell line may be established if appropriate cui taring conditions are met and preferably if the cell is suitably adapted for this purpose (for instance a stem cell).
  • the model can be used to study the effects of mutations on the animal or leukocyte and the leukocyte response, as well as development and/or progression of the disease using measures commonly used in the study of the disease.
  • such a model is useful for studying the effect of a putativeiy pharmaceutically active compound or gene therapy on the disease or leukocyte response.
  • a response- or disease- associated gene or polynucleotide can be modified to give rise to the aberrant response or disease in the model, and then putativeiy pharmaceutically active compound and/or gene therapy can be administered so as to observe whether the reponse or disease development and/or progression is inhibited or reduced.
  • the method comprises modifying so as to produce, one or more, advantageously 3-50 or more response- or disease-associated or correlated gene(s) or polynucleotide's).
  • a genetically modified animal or leukocyte may be compared with an animal predisposed to development of the disease, such that administering putative gene therapy, or pharmaceutically acceptable compound(s), or any combination thereof can be performed to assess how such putative therapy(ies) or treatment(s) may perform in a human.
  • the invention can involve contacting a test compound with a leukocyte or administering to a eukaryote a test compound, wherein the eukaryote or leukocyte comprises one or more, e.g., 3-50 or more mutations from the CRISPR-Cas system, e.g., in an animal that expresses Cas9 and to which RNA(s) generating the mutations has / have been administered; and detecting a reduction or an augmentation of a cell signaling event associated with the mutation(s) or lack thereof.
  • Screening of such putative pharmaceutically active compound(s) and/or gene therapy(ies) can be by cellular function change and/or intracellular signaling or extracellular signaling or change in intracellular expression or change in expression of protein on the cell surface. Such screening can involve evaluating for dosages or dose curves, as well as combinations of potential drugs and/or therapies.
  • An altered expression of one or more genome sequences associated with a signaling biochemical pathway can be determined by assaying for a difference in the mRNA levels of the corresponding genes between the disease model eukaryote or animal or leukocyte thereof and a normal eukaryote, animal, or leukocyte, and to ascertain whether when the disease model is administered or contacted with a candidate chemical agent or gene therapy it reverts to or towards normal .
  • An assay can be for mutation(s)-induced alteration in the level of mRNA transcripts or corresponding polynucleotides or polypeptides in comparison with such level (s) in a normal eukaryote or animal and whether such level(s) are placed towards or to normal when a therapy or treatment or agent is employed.
  • the invention comprehends deliver of multiple RNA(s), e.g., sg A(s), e.g., 3-50 or more, e.g., 3, 16, 32, 48, 50 or more to the transgenic Cas9 eukaryote and thereafter screening cells, tissue, tumors or the eukaruote for fibers or formation of fibers (with it understood that "eukaryote” is as herein discussed to include animal, mammal, etc).
  • Inducing multiple mutations also enables the skilled person to divine new combinations of mutations that give rise to aberrant leukocyte responses or diseases associated with leukocytes such as immune system disorders or cancer.
  • the ability to induce multiple mutations that accelerate or change the rate of a leukocyte- associated disease, e.g., an immune system disorder or cancer accordingly provides many advantages heretofore unknown in research and development of pharmaceuticals, therapies and treatments for such disorders.
  • the invention can involve leukocytes, e.g., non-human, e.g., animal, such as mammal, e.g., primate, rodent, mouse, rat, rabbit, etc., or even human leukocytes, transformed to contain Cas9, e.g., by a vector that contains nucleic acid molecule(s) encoding a Cas9, e.g., with nucleic acid(s) encoding a promoter and at least one NLS, advantageously two or more NLSs, or leukocytes that have had their genome altered, e.g., through the vector being an integrating virus or through such leukocytes being stem cells or ceils that give rise to a cell line or a living organism (but wherein such an organism is advantageously non-human), that contains and expresses nucleic acid molecuie(s) encoding Cas9.
  • leukocytes e.g., non-human, e.g., animal, such as ma
  • leukocytes are then transplanted into or onto an animal suitable for being a leukocyte-associated disease, e.g., immune system or cancer, model, e.g., fish, a rodent such as a mouse, chickens or chicken embryo or chicken embryo membrane.
  • a leukocyte-associated disease e.g., immune system or cancer
  • model e.g., fish
  • rodent such as a mouse
  • chickens or chicken embryo or chicken embryo membrane e.g., fish
  • the leukocytes proliferate on or in the non-human eukaryote, e.g., animal model.
  • RNA(s) or vector(s) e.g., AAV, adenovirus, lentivirus containing or providing RNA(s), e.g., under the control of a promoter such as a U6 promoter and/or paiticle(s) and/or nanopaiticle(s) containing the RNA(s) and/or vector(s), whereby the RNA(s) direct the Cas9 in the cells to provide mutation, e.g., a plurality of r itation(s) such as from 3 to 30 mutations, advantageously mutation(s) associated or correlated with a a leukocyte-associated disease, whereby the non-human eukaryote, e.g., animal model is transformed into being a non-human eukaryote, e.g., animal model for the
  • the non-human eukaryote e.g., animal model can then be used for testing, e.g., as to potential therapy and/or putative treatment via a possibly pharmaceutically active compound.
  • the administering can be at or to or for body deliver ⁇ ' to the proliferated heterologous transplanted Cas9-containing leukocytes, e.g., direct injection at or near such proliferated heterologous transplanted Cas9-containing leukocytes, or injection or other administration in such a way that the RNA(s) are delivered into the proliferated heterologous transplanted Cas9- containing leukocytes, e.g., injection into the bloodstream whereby bodily functions transport to the proliferated heterologous transplanted Cas9-containing leukocytes.
  • barcoding techniques of WO/2013/138585 Al can be adapted or integrated into the practice of the invention.
  • WO/2013/138585 Al provides methods for simultaneously determining the effect of a test condition on viability or proliferation of each of a plurality of genetically heterogeneous cell types.
  • the methods include: providing a unitary sample comprising a plurality of, e.g., five, ten, twenty, twenty-five, or more, genetically heterogeneous cell types (each individual cell type is genetically homogeneous within itself, but differs from the others in the plurality), wherein each cell type further comprises: (i) an exogenous nucleic acid tag stably integrated into the genome of the cells, e.g., a tag comprising a core sequence that is unique to each cell type, and flanking amplification primer binding sequences that are the same in all of the ceils of the plurality, and (ii) optionally, a marker, e.g., a selectable or detectable marker; and a known number of cells of each cell type is present in the sample; exposing the sample to a test condition for a selected time; and detecting a level of the exogenous nucleic acid tag in each cell type, wherein the level of the exogenous nucleic acid tag is proportional to the number of living
  • WO/2013/138585 Al also provides methods for simultaneously determining the effect of a test condition on viability or proliferation of each of a plurality of genetically heterogeneous cell types, wherein the methods include providing a unitary sample comprising a plurality of, e.g., five, ten, twenty, twenty-five, or more, genetically heterogeneous cell types, wherein each cell type further comprises: (i) an exogenous nucleic acid tag stably integrated into the genome of the cells, e.g., comprising a core sequence that is unique to each cell type, and flanking amplification primer binding sequences that are the same in all of the cells of the plurality, and (ii) optionally, a selectable or detectable marker; and a known number of cells of each cell type is present in the sample; implanting the sample into a living animal; exposing the exogenous nucleic acid tag stably integrated into the genome of the cells, e.g., comprising a core sequence that is unique to each cell type,
  • the number of living cells in the sample after exposure to the test condition as compared to the reference number of cells indicates the effect of the test condition on viability or proliferation of each cell type.
  • the tag can be Cas9 or another TAG or marker that is integrated into the genome of ceils to be transplanted into or onto a non-human eukaryote, e.g., animal model, or that is integrated into the genome of the non- human transgenic eukaryote, e.g., animal, mammal, primate, rodent, mouse, rat, rabbit, etc (along with coding for Cas9).
  • the test condition can be the administration or delivery of the RNA(s) to guide the Cas9 to induce one or more or a plurality, e.g., 3-50 or more, mutations.
  • the test condition can be the administration, delivery or contacting with a putative chemical agent treatment and/or gene therapy treatment.
  • the tag can also be the one or more or a plurality, e.g., 3-50 or more mutations, and the test condition can be the administration, delivery or contacting with a putative chemical agent treatment and/or gene therapy treatment.
  • the invention comprehends delivering the C ISP -Cas system and/or component(s) thereof to the transgenic non-human eukaryote, e.g., mammal; for instance, to leukocytes of a mammal, e.g., a non-human mammal.
  • the transgenic non-human eukaryote e.g., mammal
  • leukocytes of a mammal e.g., a non-human mammal.
  • delivery of the CRISPR-Cas system and/or component(s) thereof and/or coding for components) thereof can be as in WO 2014/093622 (PCT/US2013/074667), with AAV, lentivirus, and adenovirus vectors or particles or nanoparticles (including particle bombardment techniques that can include gold or other elemental, e.g., tungsten particles) preferred, and use thereof in the practice of the invention is within the ambit of the skilled artisan from this disclosure and the knowledge in the art, e.g., WO 2014/093622 (PCT/US2013/074667), incorporated herein by reference.
  • the invention involves at least one component of the CRISPR.
  • the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host leukocyte.
  • the invention further provides leukocytes produced by such methods, and animals comprising such leukocytes.
  • a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to a leukocyte.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian leukocytes.
  • Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoiiposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, cell-penetrating peptides and agent-enhanced uptake of DNA.
  • Cell- penetrating peptides are described in, for example, Stalmans et al. PLoS One. 2013; 8(8): e71752 and MiUetti et al. (2012), Dmg Discov Today 17: 850-860.
  • Lipofection is described in e.g., U.S. Pat. Nos.
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91 /16024, Delivery can be to leukocytes in culture (e.g. in vitro or ex vivo administration) or leukocytes in an animal (e.g. in vivo administration).
  • lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes
  • the preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et a!., Cancer Gene Then 2:291-297 (1995); Behr et al, Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al, Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.
  • R A or DN.A viral based systems for the deliver ⁇ ' of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat leukocytes in vitro, and the modified leukocytes may optionally be administered to patients (ex vivo).
  • Conventional viral based systems could include retroviral, lenti virus, adenoviral, adeno-associated and herpes simplex vims vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different ceil types and target tissues.
  • Adeno-associated vims may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al, Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641 ; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or ⁇ 3 1 7 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producer a cell line that packages a nucleic acid vector into a viral particle.
  • the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucieotide(s) to be expressed.
  • the missing viral functions are typically supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a ceil line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAA'' genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Accordingly, AAV is considered an ideal candidate for use as a transducing vector.
  • AAV transducing vectors can comprise sufficient cis-acting functions to replicate in the presence of adenovirus or herpesvirus or poxvirus (e.g., vaccinia vims) helper functions provided in trans.
  • Recombinant AAV rAAV
  • the AAV cap and/or rep genes are deleted from the viral genome and replaced with a DNA segment of choice.
  • Current AAV vectors may accommodate up to 4300 bases of inserted DNA.
  • the invention provides rAAV and methods for preparing rAAV. For example, plasmid(s) containing or consisting essentially of the desired viral construct are transfected into AAV-infected cells. In addition, a second or additional helper plasmid is cotransfected into these cells to provide the AAV rep and/or cap genes which are obligator ⁇ ' for replication and packaging of the recombinant viral construct.
  • rAAV is harvested. Traditionally rAAV is harvested from the cells along with adenovirus. The contaminating adenovirus is then inactivated by heat treatment. In the instant invention, rAAV is advantageously harvested not from the cells themselves, but from cell supernatant.
  • rAAV can be prepared by a method that comprises or consists essentially of: infecting susceptible cells with a rAAV containing exogenous DNA including DNA for expression, and helper virus (e.g., adenovirus, herpesvirus, poxvirus such as vaccmia virus) wherein the rAAV lacks functioning cap and/or rep (and the helper virus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus) provides the cap and/or rev function that the rAAV lacks); or infecting susceptible cells with a rAAV containing exogenous DNA including DNA for expression, wherein the recombinant lacks functioning cap and/or rep, and traiisfecting said ceils with a plasmid supplying cap and/or rep function that the rAAV lacks; or infecting susceptible cells with a rA
  • helper virus e.g., adenovirus, herpesvirus, poxvirus such
  • the rAAV can be from an AAV as herein described, and advantageously can be an rAA l , rAAV2, AAV5 or rAAV having hybrid or capsid which may comprise AAVl, AAV2, AAV5 or any combination thereof.
  • other cells that can be used in the practice of the invention and the relative infectivity of certain AAV serotypes in vitro as to these ceils; see Grimm, D, et ai, J. Virol. 82: 5887-5911 (2008).
  • the invention provides rAAV that contains or consists essentially of an exogenous nucleic acid molecule encoding a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system or component(s) or coding therefor, e.g., a plurality of cassettes comprising or consisting a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CR!SPR-associated (Cas) protein (putative nuclease or helicase proteins), e.g., Cas9 and a terminator, and a two, or more, advantageously up to the packaging size limit of the vector, e.g., in total (including the first cassette) five, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNAI -terminator, Promoter- gRNA2-
  • Promoter-gR A(N Herminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector), or two or more individual rAAVs, each containing one or more than one cassette of a CRISPR system, e.g., a first rAAV containing the first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding Cas, e.g., Cas9 and a terminator, and a second rAAV containing a plurality, four, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRN A 1 -terminator, Promoter-gRN A2-terminator ...
  • gRNA nucleic acid molecule encoding guide RNA
  • Promoter-gRN A(N) ⁇ termmator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector).
  • N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector.
  • the promoter is in some embodiments advantageously human Synapsin I promoter (hSyn).
  • a leukocyte transiently transfected with the components of a CRJSPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new leukocyte cell line comprising leukocytes containing the modification but lacking any other exogenous sequence.
  • leukocytes transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
  • a Cas9-expressing model provided for herein is used, then only delivery of guide(s) is necessary.
  • one or more vectors described herein are used to produce a non-human transgenic Cas9 eukaryote, e.g., animal, mammal, primate, rodent, mouse, rat, rabbit.
  • the transgenic animal is a mammal, such as a mouse, rat, or rabbit.
  • Guides or RNA(s) can be delivered via the same vector types as Cas9.
  • both guides or R A(s) and Cas9 are being delivered a dual-vector system where the Cas9 is delivered via in vivo expression from an AAV vector and the guide(s) are delivered by a separate AAV vector. This can be done substantially contemporaneousl (i.e. co-delivery), but it could also be done at separate points in time, separated even by weeks or months. Of course, the ultimate separation is where the transgenic Cas9 eukaryote is generated and thereafter the guide(s) or RNA(s) are delivered. Alternatively a first round of CRISPR-Cas9 systems can be delivered, and subsequently further guides or RNA(s) are delivered as the original Cas9 is still functional in the target cells may be re-used. If the Cas9 is under the control of an inducible promoter, then induction of transcription of new CAs9 in the target cells is preferred.
  • Aerosolized delivery is preferred for AAV or adenovirus delivery in general.
  • An adenovirus or an AAV particle may be used for deliver ⁇ '.
  • Suitable gene constructs, each operably linked to one or more regulatory sequences, may be cloned into the delivery vector.
  • Cbh or EFla promoter for Cas9, U6 or HI promoter for chimeric guide RNA may be advantageous.
  • Cas9 and/or RNA(s) can be delivered using particles, adeno associated vims (AAV), lentivirus, adenovirus or other piasmid or viral vector types, in particular, using formulations and doses from, for example, US Patents Nos.
  • the route of administration, formulation and dose can be as in US Patent No 5,846,946 and as in clinical studies involving plasmids. Doses may be based on or extrapolated to an average 70 kg individual, and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the vectors can be injected into the tissue of interest.
  • the expression of Cas9 can be driven by a cell-type specific promoter,
  • vectors that may be used in the practice of the invention, integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors (and hence both lentiviral and retroviral vectors may be used in the practice of the invention). Moreover, lentiviral vectors are preferred as they are able to transduce or infect non-dividing cells and typically produce high viral titers.
  • Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors that may be used in the practice of the invention include those based upon murine leukemia vims (MuLV), gibbon ape leukemia virus (GaLV), Simian Immune deficiency vims (8IV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al, J.
  • a minimal non-primate lentiviral vector such as a lentiviral vector based on the equine infectious anemia virus (EIAV) (see, e.g., Balagaan, J Gene Med 2006; 8: 275 - 285, Published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: lQ,10Q2/jgm.845).
  • the vectors may have cytomegalovirus (CMV) promoter driving expression of the target gene.
  • CMV cytomegalovirus
  • Intracameral, subretinal, intraocular and intravitreal injections are all within the ambit of the instant invention (see, e.g., Balagaan, J Gene Med 2006; 8: 275 - 285, Published online 21 November 2005 in Wiley InterScience (wvvw.interscieiice.vviiey.com).
  • RetinoStat® an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostain and angiostatin that is delivered via a subretinal injection for the treatment of the web form of age-related macular degeneration is also contemplated (see, e.g., Binley et al, HUMAN GENE THERAPY 23:980-991 (September 2012)). Such a vector may be modified for practice of the present invention.
  • RetinoStat® e.g., 1.1 x 10 5 transducing units per eye (TU/eye) in a total volume of 100 ⁇
  • TU/eye transducing units per eye
  • the invention also can be practiced with an adenovirus vector, e.g., an E1-, partial E3-, E4-deleted adenoviral vector may be used in the practice of the invention.
  • an adenovirus vector e.g., an E1-, partial E3-, E4-deleted adenoviral vector may be used in the practice of the invention.
  • Such vectors are safe as twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) were given a single mtravitreous injection of an EI-, partial E3-, E4-deleted adenoviral vector expressing human pigment epithelium-derived factor (AdPEDF.ll) (see, e.g., Campoehiaro et al..
  • Adenoviral vector-mediated RNA transfer appears to be a viable approach for delivery of NA(S).
  • NSS normal saline solution
  • siRNA For siRNA, a rat was injected into the great saphenous vein with 12.5 ug of a siRNA and a primate was injected injected into the great saphenous vein with 750 ⁇ g of a siRNA. This can be adapted to or extrapolated from in the practice of the present invention.
  • viral vectors including retroviral vectors, lentiviral vectors, adenovirus vectors, or AAV vectors.
  • a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified according to diameter. Coarse particles cover a range between 2,500 and 10,000 nanometers. Fine particles are sized between 100 and 2,500 nanometers. Uftrafine particles, or nanoparticles, are generally between 1 and 100 nanometers in size.
  • a particle delivery system/formulation is defined as any biological delivery system/formulation which includes a particle in accordance with the present invention.
  • a particle in accordance with the present invention is any entity having a greatest dimension (e.g. diameter) of less than 100 microns ( ⁇ ).
  • inventive particles have a greatest dimension of less than 10 ⁇ .
  • inventive particles have a greatest dimension of less than 2000 nanometers (nm).
  • inventive particles have a greatest dimension of less than 1000 nanometers (nm).
  • inventive particles have a greatest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.
  • inventive particles have a greatest dimension (e.g., diameter) of 500 nm or less.
  • inventive particles have a greatest dimension (e.g., diameter) of 250 nm or less, in some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 200 nm or less.
  • inventive particles have a greatest dimension (e.g., diameter) of 150 nm or less.
  • inventive particles have a greatest dimension (e.g., diameter) of 100 nm or less. Smaller particles, e.g., having a greatest dimension of 50 nm or less are used in some embodiments of the invention. In some embodiments, inventive particles have a greatest dimension ranging between 25 nm and 200 nm. Particle characterization (including e.g., characterizing morphology, dimension, etc.) is done using a variety of different techniques.
  • TEM electron microscopy
  • SEM atomic force microscopy
  • AFM dynamic light scattering
  • XPS X-ray photoelectron spectroscopy
  • XRD powder X-ray diffraction
  • FTIR Fourier transform infrared spectroscopy
  • MALD I-TOF matrix-assisted laser desorption/ionization time-of-fli ght mass spectrometry
  • NMR nuclear magnetic resonance
  • Characterization may be made as to native particles (i.e., preloading) or after loading of the cargo (herein cargo refers to e.g., one or more components of CRISPR- Cas system e.g., CRISPR enzyme or mRNA or guide R.NA, or any combination thereof, and may include additional carriers and/or excipients) to provide particles of an optimal size for delivery for any in vitro, ex vivo and/or in vivo application of the present invention.
  • particle dimension (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS).
  • DLS dynamic laser scattering
  • Particles deliver ⁇ ' systems within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles.
  • any of the delivery systems described herein including but not limited to, e.g., lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene gun may be provided as particle delivery systems within the scope of the present invention.
  • nanop article refers to any particle having a diameter of less than 1000 nm.
  • nanoparticles of the invention have a greatest dimension (e.g., diameter) of 500 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 25 nm and 200 nm.
  • nanoparticles of the invention have a greatest dimension of 100 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 35 nm and 60 nm.
  • Nanoparticles encompassed in the present invention may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof.
  • Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles).
  • Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drag carriers or imaging agents and may be adapted for similar purposes in the present invention.
  • Nanoparticles with one half hydrophiiic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self- assemble at water/oil interfaces and act as solid surfactants. Doses of about 5 me/kg are contemplated, with single or multiple doses, depending on the target tissue. It is mentioned herein experiments involving mice involve 20g mammals and that dosing can be scaled up to a 70 kg human.
  • nanoparticles that can deliver RNA see, e.g., Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug 6;110(32): 12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar 13;13(3):1059-64; Karagiannis et al, ACS Nano. 2012 Oct 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug 28;6(8):6922-9 and Lee et al, Nat Nanotechnol. 2012 Jun 3;7(6):389-93.
  • Lipid Nanoparticles, Spherical Nucleic Acid (SNATM) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplate as a means for delivery of CRISPR/Cas system or component(s) thereof or vector(s) to intended targets.
  • Particles, nanoparticles, and the like and vectors are advantageous for delivering the RNA(s) of the CRISPR-Cas9 system and particles and nanoparticles and the like may be advantageous for delivery of vector containing nucleic acid(s) encoding or comprising RNA(s) of the invention.
  • RNA(s) also herein sometimes termed " ' guides"
  • the Cas9 may be delivered via a viral vector or be constitutively or inducibly or conditionally expressed and that guides specific to genomic targets are delivered separately.
  • the 7C1 nanoparticle can be formulated to mediate delivery of sgRNA to constitutive!' active Cas9 mouse. Synthesis of the 7C1 nanoparticle is described in Dahimaii et al., 2014, Nature Nanotechnoiogy.
  • kits containing any one or more of the elements discussed herein. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language. In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g.
  • a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof.
  • the buffer is alkaline.
  • the buffer has a pH from about 7 to about 10.
  • the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.
  • the kit comprises a homologous recombination template polynucleotide.
  • the kit comprises one or more of the vectors and/or one or more of the polynucleotides described herein.
  • the kit may advantageously allow the provision of all elements of the systems of the invention.
  • Kits can involve vector(s) and/or particle(s) and/or nanoparticle(s) containing or encoding RNA(s) for 3-50 or more mutations to be administered to a non-human transgenic Cas9 eukaryote, e.g., animal, mammal, primate, rodent, etc., with such a kit including instructions for administering to such a eukaryote; and such a kit can optionally include a transgenic Cas9 eukaryote, or when such a transgenic Cas9 eukaryote has Cas9 expression that is inducible or conditional, e.g., Cre-dependent, the kit may also include a mate that expresses the compound that induces or is the trigger or condition for Cas9 expression,
  • nucleic acids, amino acids and proteins The invention uses nucleic acids to bind target DNA sequences. This is advantageous as nucleic acids are much easier and cheaper to produce than proteins, and the specificity can be varied according to the length of the stretch where homology is sought. Complex 3-D positioning of multiple fingers, for example is not required.
  • polynucleotide “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown.
  • the following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • a "wild type” can be a base line.
  • variant should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.
  • non-naturaily occurring or “engineered” are used interchangeably and indicate the involvement of the hand of man.
  • nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types, A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantial!)' does not hybridize to no -target sequences.
  • Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
  • Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology- Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y.
  • complementary or partially complementary sequences are also envisaged. These are preferably capable of hybridising to the reference sequence under highly stringent conditions.
  • relatively low-stringency hybridization conditions are selected: about 20 to 25° C lower than the thermal melting point (T m ).
  • T m is the temperature at which 50% of specific target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH.
  • highly stringent washing conditions are selected to be about 5 to 15° C lower than the T m .
  • moderately-stringent washing conditions are selected to be about 15 to 30° C lower than the T m .
  • Highly pennissive (very low stringency) washing conditions may be as low as 50° C below the T m , allowing a high level of mis-matching between hybridized sequences.
  • Other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of homology between target and probe sequences.
  • Preferred highly stringent conditions comprise incubation in 50% formamide, 5 X SSC, and 1 % SDS at 42° C, or incubation in 5xSSC and 1% SDS at 65° C, with wash in 0.2xSSC and 0.1% SDS at 65° C.
  • “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PGR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the "complement" of the given sequence.
  • the term "genomic locus” or “locus” is the specific location of a gene or DNA sequence on a chromosome.
  • a “gene” refers to stretches of DNA or RNA that encode a polypeptide or an RNA chain that has functional role to play in an organism and hence is the molecular unit of heredity in living organisms.
  • genes include regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
  • a gene includes, but is not necessarily limited to, promoter sequences, terminators, transiationai regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
  • expression of a genomic locus or “gene expression” is the process by which information from a gene is used in the synthesis of a functional gene product.
  • the products of gene expression are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is functional RNA.
  • the process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and arciiaea) and viruses to generate functional products to survive.
  • expression of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context.
  • expression also refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA. or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as "gene product,” if the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotie cell.
  • the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, giycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • domain or “protein domain” refers to a part of a protein sequence that may exist and function independently of the rest of the protein chain.
  • sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.
  • the capping region of the dTALEs descri bed herein have sequences that are at least 95% identical or share identity to the capping region amino acid sequences provided herein. Sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA, etc.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S. A; Devereux et ai., 1984, Nucleic Acids Research 12:387).
  • Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al,, 1999 ibid - Chapter 18), FASTA (Atschul et al, 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
  • Percentage (%) sequence homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion may cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed.
  • sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology or identity score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology or identity.
  • these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment wit as few gaps as possible - reflecting higher relatedness between the two compared sequences - may achieve a higher score than one with many gaps.
  • “Affinity gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system.
  • High gap penalties may, of course, produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore first requires the production of an optimal alignment, taking into consideration gap penalties.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984 Nuc. Acids Research 12 p387).
  • Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4 th Ed. - Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett.
  • % homology may be measured in terms of identity
  • the alignment process itself is typically not based on an all-or- nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table, if supplied (see user manual for further details).
  • the public default values for the GCG package or in the case of other software, the default matrix, such as BLOSUM62.
  • percentage homologies may be calculated using the multiple alignment feature in DNAS1S ' M (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73( 1), 237-244). Once the software has produced an optima! alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
  • the sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups, Amino acids may be grouped together based on the properties of their side chains alone. However, it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of a Venn diagram (Livingstone CD. and Barton G.J. (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput. Appl. Biosci.
  • Embodiments of the invention include sequences (both polynucleotide or polypeptide) which may comprise homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue or nucleotide, with an alternative residue or nucleotide) that may occur i.e., like-for-like substitution in the case of amino acids such as basic for basic, acidic for acidic, polar for polar, etc.
  • Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenyl glycine.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine pyriylalanine
  • thienylalanine naphthylalanine
  • phenyl glycine unnatural amino acids
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ -alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ -alanine residues.
  • a further form of variation which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the ⁇ -carbon substitueiit group is o the residue's nitrogen atom rather than the ⁇ -carbon.
  • amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity.
  • Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGoldTM, T7 DNA polymerase, lenow fragment of E.coli DNA polymerase, and reverse transcriptase.
  • a preferred amplification method is PGR.
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector wherein viraily-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively- linked. Such vectors are referred to herein as "expression vectors.”
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of lasm ids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host ceil when the vector is introduced into the host ceil).
  • aspects of the invention relate to bicistronic vectors for chimeric RNA and Cas9.
  • Bicistronic expression vectors for chimeric RNA and Cas9 are preferred.
  • Cas9 is preferably driven by the CBh promoter.
  • the chimeric RNA may preferably be driven by a Pol III promoter, such as a U6 promoter. Ideally the two are combined.
  • the chimeric guide RNA typically consists of a 20bp guide sequence (Ns) and this may be joined to the tracr sequence (running from the first "U" of the lower strand to the end of the transcript). The tracr sequence may be truncated at various positions as indicated.
  • the guide and tracr sequences are separated by the tracr-mate sequence, which may be GUUUUAGAGCUA. This may be followed by the loop sequence GAAA as shown. Both of these are preferred examples.
  • Applicants have demonstrated Cas9-mediated indels at the human EMXl and FVALB loci by SURVEYOR assays.
  • ChiRNAs are indicated by their "+n" designation, and crRNA refers to a hybrid RNA where guide and tracr sequences are expressed as separate transcripts.
  • chimeric RNA may also be called single guide, or synthetic guide RNA (sgRNA).
  • the loop is preferably GAAA, but it is not limited to this sequence or indeed to being only 4bp in length.
  • preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences.
  • the sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG.
  • a suitable vector can be introduced to a cell or an embryo via one or more methods known in the art, including without limitation, microinjection, eiectroporation, sonoporation, biolistics, calcium phosphate-mediated transfection, catiomc transfection, liposome transfection, dendnmer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impaiefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.
  • the vector is introduced into an embryo by microinjection.
  • the vector or vectors may be microinjected into the nucleus or the cytoplasm of the embryo.
  • the vector or vectors may be introduced into a cell by nucieofection.
  • regulatory element is intended to include promoters, enhancers, internal ribosomaf entry sites ( IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • IRES internal ribosomaf entry sites
  • regulatory elements e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences.
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host ceil and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • a tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific.
  • a vector comprises one or more pol III promoter (e.g. 1 , 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g.
  • pol III promoters include, but are not limited to, U6 and HI promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma vims (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41 :521-530 (1985)], the SV40 promoter, the dihydro folate reductase promoter, the ⁇ -actin promoter, the phosphoglyceroi kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma vims
  • CMV cytomegalovirus
  • PGK phosphoglyceroi kinase
  • enhancer elements such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc. Natl. Acad. Sci. US A., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed , the level of expression desired, etc.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Vectors can be designed for expression of CRISPR transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells.
  • CRISPR transcripts e.g. nucleic acid transcripts, proteins, or enzymes
  • CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Vectors may be introduced and propagated in a prokaryote or prokaryotic cell.
  • a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system).
  • a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.
  • Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein.
  • Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
  • E. coli expression vectors include pTrc (Araranti et al., (1988) Gene 69:301-315) and pET l id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • a vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSecl (Baidari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933- 943), pJRY88 (Schultz et al., 1987. Gene 54: 1 13-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif.).
  • a vector drives protein expression in insect cells using baculovirus expression vectors.
  • Bacuiovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al., 1983. Mol. Cell, Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).
  • the expression vector's control functions are typically provided by one or more regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver- specific; Pinkert, et al., 1987, Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
  • a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • SPIDRs Sacer Interspersed Direct Repeats
  • SSRs interspersed short sequence repeats
  • the CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al, OMICS J. Integ. Biol, 6:23-33 [2002]; and Mojica et al, Mol. Microbiol., 36:244-246 [2000]).
  • SRSRs short regularly spaced repeats
  • the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al, [2000], supra).
  • CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol.
  • the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme).
  • a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glueuronldase, iuciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • beta-glueuronldase beta-glueuronldase
  • iuciferase iuciferase
  • GFP green fluorescent protein
  • HcRed HcRed
  • DsRed
  • a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence,
  • a CRISPR enzyme may form a component of an inducible system.
  • the inducible nature of the system would allow for spatiotemporal control of gene editing or gene expression using a form of energy.
  • the form of energy may include but is not limited to electromagnetic radiation, sound energy, chemical energy and thermal energy.
  • inducible system examples include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytoehrorne, LOV domains, or cryptochrome).
  • the CRISPR enzyme may be a part of a Light Inducible Transcriptional Effector (LITE) to direct changes in transcriptional activity in a sequence-specific manner.
  • the components of a light may include a CRISPR enzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsis thaliana), and a transcriptional activation/repression domain.
  • LITE Light Inducible Transcriptional Effector
  • Embodiments of the present invention involve modulating leukocyte activity, as well as identifying genes associated with leukocyte responses and modeling of aberrant leukocyte activation and diseases associated with leukocytes.
  • the leukocyte(s) may be any subtype or population of leukocytes, e.g. dendritic cells, monocytes, macrophages, lymphocytes (e.g. T cells or B cells or Natural Killer cells), neutrophils, eosinophils, or basophils, or any combination or subgroup of the above.
  • Precursors of mature leukocytes are also encompassed by the term leukocytes, e.g. hematopoietic stem and progenitor cells.
  • Leukocyte subtypes may be identified by characteristic morphology and/or cell surface markers, as is well known in the art,
  • the leukocyte(s) may comprise a subtype of dendritic cells, monocytes, macrophages, lymphocytes (e.g. T cells or B cells or Natural Killer cells), neutrophils, eosinophils, or basophils, e.g. defined by one or more cell surface markers.
  • the leukocytes comprise bone marrow-derived dendritic cells (BMDCs).
  • the leukocytes may comprise helper, cytotoxic, memory and/or regulatory T cells, e.g. CD4+, CD8+, CD45RO+ and/or Foxp3+ T cells.
  • various types of leukocyte response may be modeled or investigated, e.g. depending on the type of leukocyte or subtype thereof.
  • the leukocyte response may comprise activation, inhibition, migration, exhaustion, adhesion, differentiation and/or proliferation.
  • the leukocytes are dendritic cells and the response comprises activation.
  • dendritic ceils may be activated by stimulation with bacterial Hpopo!ysaeeharide (LPS), in some embodiments dendritic cell activation may be measured by determining expression of one or more products or cell surface markers associated with dendritic cell activation, e.g. tumor necrosis factor alpha (TNF-a) or CD86.
  • TNF-a tumor necrosis factor alpha
  • Activation of dendritic cells may be suppressed by anti-inflammatory cytokines such as interleukin-10.
  • stimulation of dendritic cells with LPS in the presence of lL-10 allows the identification of mutations that overcome the suppression of dendritic cell activation by lL-10.
  • the leukocytes are T cells and the response comprises differentiation, for instance towards a Thl, Th2, Treg or Thl 7 phenotype, or maintenance of each phenotype.
  • Differentiation of T cells towards particular phenotypes may be stimulated by specific cytokines.
  • interleukin-12 IL-12
  • interleukin-4 IL-4
  • TGFp transforming growth factor beta
  • IL-6 IL-6
  • Differentiation towards particular responses may be measured by determining expression of particular markers characteristic of each response type.
  • interferon-)' IFN ⁇
  • IL-4 may be measured to indicate a Th2 response
  • TGFP may be measured to indicate a T reg response
  • IL-17 may be measured to indicate a Thl 7 response.
  • the T cells are from a transgenic mouse having cells thai- express Cas9 and an ovalbumin-specific ⁇ - ⁇ cell receptor.
  • Such transgenic mice may be obtained, for example, by crossing Cas9 transgenic mice as described above (e.g. having a Cas9 transgene knocked into the Rosa26 locus) with e.g. OT-II transgenic mice.
  • OT-II transgenic mice express the mouse alpha-chain and beta-chain T cell receptor that pairs with the CD4 coreceptor and is specific for chicken ovalbumin 323-339 in the context of I- A b.
  • OT-I I mice there is a four-fold increase in the CD4 to CDS peripheral T cell ratio, and lymph node T cells demonstrate a dose-dependent proliferative response to the specific ovalbumin ligand.
  • OT-II mice are described, for example, in Barnden el al. (1998) Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements, Immunol Cell Biol 76(l):34-40, incorporated by reference.
  • a decreased response in a leukocyte is typically indicative that a gene mutated in the leukocyte by delivery of the vector is a positive regulator of the response. For instance, where delivery of the vector introduces one or more loss- of-function mutations into a gene that normally promotes the response, the response is expected to be decreased. Conversely an increased response in a leukocyte is typically indicative that a gene mutated in the leukocyte by delivery of the vector is a negative regulator of the response, i.e. delivery of the vector introduces one or more loss-of-function mutations into a gene expression that normally suppresses the response.
  • Embodiments of the present invention may involve modeling of any of the above responses, and in particular aberrant leukocyte responses.
  • aberrant leukocyte responses it is typically meant a leukocyte response that is deficient or excessive, for instance deficient or excess activation, differentiation or proliferation.
  • leukocytes in which that gene is inactivated may be used to model such aberrant responses.
  • dendritic cells in which the gene contains one or more loss-of-function mutations may ⁇ be used as a model of deficient dendritic cell activation.
  • Such a model may be used to identify agents which promote dendritic cell activation, e.g. for use in immunotherapy for cancer.
  • inventions may involve modeling diseases associated with leukocytes.
  • diseases associated with leukocytes it is typically meant any disorder or condition in which leukocytes or a subtype thereof influence the disease pathology, especially conditions where deficient or excessive leukocyte responses are associated with the disease.
  • Leukocyte-associated diseases include, e.g. immune system disorders and cancer.
  • the disease may be an autoimmune disease, a chronic persistent infection, leukemia, leukopenia or transplant rejection.
  • autoimmune diseases involve the generation of an immunogenic response against one or more self tissues or cells.
  • autoimmune diseases include rheumatoid arthritis, multiple sclerosis, lupus erythematosis, myasthenia gravis, scleroderma, Crohn's disease, ulcerative colitis, Hashimoto's disease, Graves' disease, Sjogren's syndrome, Addison's disease, insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease and thyroiditis.
  • IDDM insulin-dependent diabetes mellitus
  • Infections may be considered to be chronic and persistent if, for example, the infection persists for at least 3 months, more preferably at least 6 months.
  • Examples of chronic persistent infections include tuberculosis, the hepatitis B, hepatitis C or HIV.
  • Transplant rejection may be either chronic or acute, including rejection of allogeneic or xenogeneic transplants.
  • the disease may also be graft-versus-host disease, i.e. in which leukocytes from a donor attack cells of a transplant recipient.
  • Cancer may be defined as a condition involving uncontrolled cell growth.
  • the models of the present invention may be used to model the aberrant leukocyte responses occurring in such diseases. For instance, excessive leukocyte responses may underlie the pathology in conditions such as autoimmune diseases and transplant rejection.
  • Targeting a negative regulator of leukocyte responses e.g. the mouse A20 locus in dendritic cells, see Example 2 may serve as a model for such excessive leukocyte responses.
  • the suppression of immune responses against tumor antigens is important in the pathogenesis of many types of cancer.
  • Targeting a positive regulator of leukocyte responses e.g. the mouse MyD88 locus in dendritic ceils, see Example 2 may be used to model deficient leukocyte responses in conditions such as cancer.
  • the method of the present invention involves delivering a plurality of different vectors, each expressing different guide RNAs, to a plurality of leukocytes, in this way, a plurality of different genes expressed in the leukocytes may be targeted, in order to screen for genes invol ved in leukocyte responses.
  • the method may involve delivering at least 100, at least 1000 or at least 10,000 vectors, wherein each vector expresses one or more guide RNAs. For instance, at least 100, at least 1000, at least 10,000 or at least 100,000 guide RNAs may be expressed in the method.
  • the method involves a genome-wide screen, e.g. in which all genes in the genome of the leukocytes are targeted.
  • a library of vectors is delivered to the leukocytes, each vector in the library expressing one or more RNAs targeted to a gene in a genome of the leukocytes, and wherein the library comprises at least one vector expressing an RNA targeted to each gene in the genome of the leukocytes.
  • the library comprises more than one RNA targeted to each gene in the genome, e.g. the library comprises two, three, four, five or six or more RNAs targeted to each gene in the genome.
  • the library may comprise, for example, at least 10,000, at least 50,000 or at least 100,000 guide RNAs.
  • the libraiy comprises a genome-wide lentiviral sgRNA library as described in Sanjana et al. (2014), Improved vectors and genome -wide libraries for CRISPR screening, Nature Methods ! 1 , 783-784, incorporated herein by reference.
  • pCAG is the promoter
  • NLS is a nuclear localization signal
  • P2A is the peptide cleavage sequence
  • EGFP is enhanced green fluorescent protein
  • WPRE is the woodchuck hepatitis virus posttranscriptional regulatory element
  • bGHpoiyA is the bovine growth hormone poly-A signal sequence.
  • the conditional version has one additional stop cassette element, loxP-SV40 poly A x3-loxP, after the promoter and before NLS-Cas9-NLS (i.e. pCAG-loxP-SV40polyAx3-loxP-NLS-Cas9-NLS-P2A-EGFP-WPRE- bGHpolyA).
  • the constitutive construct should be expressed in all cell types throughout development, whereas, the conditional construct will only allow Cas9 expression when the same ceil is expressing the Cre recombinase. This latter version will allow for tissue specific expression of Cas9 when Cre is under the expression of a tissue specific promoter. Moreover, Cas9 expression could be induced in adult mice by putting Cre under the expression of an inducible promoter such as the TET on or off system.
  • Each plasmid was functionally validated in three ways: 1) transient transfection in 293 cells followed by confirmation of GFP expression; 2) transient transfection in 293 cells followed by immunofluorescence using an antibody recognizing the P2A sequence; and 3) transient transfection followed by Surveyor nuclease assay.
  • the 293 cells may be 3 I or 293 T cells depending on the cells that are of interest.
  • the ceils are 293FT ceils. The results of the Surveyor were run out on the top and bottom row of the gel for the conditional and constitutive constructs, respectively. Each was tested in the presence and absence of chimeric RNA targeted to the hEMXl locus (chimeric RNA hEMXl.1).
  • Transgenic Cas9 mouse To generate transgenic mice with constructs, Applicants inject pure, linear DNA into the pronucleus of a zygote from a pseudo pregnant CB56 female. Founders are identified, genotyped, and baekcrossed to CBS 7 mice. The constructs were successfully cloned and verified by Sanger sequencing.
  • Knock in Cas9 mouse To generate Cas9 knock in mice Applicants target the same constitutive and conditional constructs to the Rosa26 locus. Applicants did this by cloning each into a Rosa26 targeting vector with the following elements: Rosa26 short homology arm - constitutive/conditional Cas9 expression cassette - pPG -Neo-Rosa26 long homology arm - pPGK-DTA.
  • pPGK is the promoter for the positive selection marker Neo, which confers resistance to neomycin, a 1 kb short arm, a 4.3 kb long arm, and a negative selection diphtheria toxin (DTA) driven by PG .
  • the two constructs were electroporated into Rl mESCs and allowed to grow for 2 days before neomycin selection was applied. Individual colonies that had survived by days 5-7 were picked and grown in individual wells. 5-7 days later the colonies were harvested, half were frozen and the other half were used for geiiotyping. Genotyping was done by genomic PCR, where one primer annealed within the donor piasmid (AttpF) and the other outside of the short homology arm (Rosa26-R) Of the 22 colonies harvested for the conditional case, 7 were positive (Left). Of the 27 colonies harvested for the constitutive case, zero were positive (Right).
  • conditional Cas9 mouse is crossed with a mouse expressing Cre under a tissue specific promoter, there should only be Cas9 in the tissues that also express Cre.
  • This approach may be used to edit the genome in only precise tissues by delivering chimeric RNA to the same tissue.
  • Example 2 Ex vivo genome editing in primary dendritic cells by lentiviral-mediated sgRNA expression
  • Bone marrow cells were collected from femora and tibiae and plated at concentration of 2xl0 5 / ' ml on non-treated tissue culture dishes in RPMI medium (Gibco, Carlsbad, CA, Invitrogen, Carlsbad, CA), supplemented with 10% FBS (Invitrogen), L- glutamine (Cellgro), penicillin/streptomycin (Cellgro), MEM non-essential amino acids (Cellgro), HEPES (Cellgro), sodium pyruvate (Cellgro), ⁇ - mercaptoethanol (Gibeo), and GM- CSF (20 ng mL; Peprotech).
  • SgRNAs targeting Myd88 and A20 were cloned into a guide-only lentiviral vector (Sanjana et al., 2014). At day 2, ceils were infected with lentiviruses encoding guide RNAs. Cells were expanded in the presence of GM-CSF. At day 7, infected cells were selected by adding puromycin (Invitrogen) at 5 .ug-'mL. At day 9, 100 ng/ml LPS (Invivogen) was added 30 min prior to protein analysis or 3 hours prior to mRNA expression profiling. Flow cytometry for GFP detection was performed with BD Accuri C6, Western blot was done using anti-Myd88 (R&D Systems AF3109) and anti-aetin (Abeam, ab6276) antibodies.
  • Applicants first verified the expression of Cas9 in bone marrow from constitutive Cas9 ⁇ expressing mice ( Figure 2). Similarly, Applicants validated Cas9 expression in many other immune cell types ( Figure 3). Two days after culturing bone marrow cells from the constitutive Cas9-expressing mice Applicants infected BMDCs with leiitivirus encoding two different sgRNAs targeting early exons of either MyDHH ( Figure 3) or A 20 ( Figure 4), two well- characterized positive and negative regulators of Toll-like receptor 4 (TLR4) signaling, respectively. At seven days post transduction Applicants activated cells with lipopolysacchari.de (LPS) and performed functional analysis ( Figure 1). Applicants found indels in 67-78% of sequencing reads (Figiare 5-6), leading to reduction in mRNA. ( Figure 7) and protein ( Figure 8).
  • LPS lipopolysacchari.de
  • Applicants establish a genome wide screen for positive and negative regulators of the LPS response in primary mouse dendritic ceils (DCs).
  • DCs primary mouse dendritic ceils
  • Bone marrow cells from Cas-9 expressing transgenic mice are infected with a library of lenti- virus that contains six guide RNAs for each mouse gene (including miRNAs), plus negative control guides that do not target any sequence in the mouse genome.
  • A. mixture of mouse primary cells is created and grown in condition that promote differentiation towards DCs. in this cell mixture each ceil contains on average one mutation in one of the targeted genes.
  • the next stage is to stimulate DCs with LPS (a bacterial component) and then isolate cells that fail to respond (indication of a mutation in a positive regulator of the LPS response) or that have a stronger than normal response (indication of a mutation in a negative regulator of the response).
  • the stimulation step is performed in the presence or absence of IL-10.
  • the readout for DC activation upon LPS is based on staining for the anti-inflammatory cytokine TNF-a or the cell surface receptor CD-86.
  • the last stage is to identify the guide RNAs present at higher levels in cells that show the desired phenotype (especially high or especially low expression of the readout marker CD-86 or TNF-a), and verify the results.
  • the method is shown schematically in Figure 1 1 .
  • OT-II mice which express the mouse alpha-chain and beta-chain T cell receptor that pairs with the CD4 coreceptor and is specific for chicken ovalbumin 323-339 in the context of I-A b, see Barnden et al. (1998), Immunol Cell Biol 76(l):34-40) are crossed with Cas9 transgenic mice to generate a transgenic mouse having cells that express Cas9 and an ovalbumi -specific ⁇ - ⁇ cell receptor.
  • T cells from the progeny of such mice are infected with a library of lenti-virus that contains six guide RNAs for each mouse gene (including miR As), plus negative control guides.
  • Guide libraries are used to knock out genes involved in T cell differentiation to 4 subsets: Thl, Th2, Thl7 and Treg.
  • T cells are sorted for high or low differentiation signal and the guides in those cells are sequenced to find positive and negative regulators of differenciation. The method is shown schematically in Figure 17.
  • Example 5 A. system for cell-autonomous, pooled genetic screens in BMDCs derived from Cas9- expressing mice
  • BMDCs bone marrow derived dendritic cells isolated from Cas9-expressing mice
  • sgRNAs short guide RNAs
  • Applicants isolated bone marrow cells from transgenic mice that constitutively express spCAS-9, transduced cells with lentivirases targeting these 3 control genes, and differentiated DCs for an additional seven days using GM-CSF (Experimental Procedures) (Piatt et al., 2014). Applicants then activated BMDCs with LPS, in the presence of Brefeldin to block Tnf secretion, and at 8 hours post-activation detected Tnf with a fluorescent antibody using flow cytometry. Compared to a non-targeting sgRNA control, sgRNAs targeting Myd88 or Tlr4 strongly reduced Tnf, whereas sgRNAs targeting Zfp36 increased Tnf ( Figure 18A).
  • Example 6 A genome-wide pooled sgRNA library screen in primary BMDCs
  • an aspect of the invention provides a more sensitive assay and/or screen for negative regulators by using lower levels of LPS.
  • Applicants devised two strategies to compare the abundance of sgRNAs between the Tnf 10 (average of two low bins) and the Tnf 111 bins.
  • Applicants calculated for each sgRNA a Z score, based on fold change of the abundance of each sgRNA between samples, after the abundance of each sgRNA was quantile normalized and averaged across replicates.
  • Applicants then derived a score per gene by averaging the Z scores for the 4 sgRNAs with the highest Z scores per gene.
  • the rankings subsequently diverge at ranks 150 and 50 for the positive and negative regulators, respectively ( Figure 23D and 23E). Note that the overlap is highly significant, but far from perfect. This is entirely expected given the extent of noise in the screen (and the corresponding estimate of 50% FDR at the 100 top ranked genes mark), and highlights the susceptibility of single ranking approaches to this noise.
  • the screen correctly identifies known regulators of cell viability, differentiation, Tnf expression and Tlr4 signaling.
  • sgRNAs against genes required for essential cell processes were depleted in pre-LPS samples compared to Input ( Figure 18C and Figure 23F), including those targeting genes previously annotated as essential (Hart et al., 2014) (p ⁇ 10 ⁇ l6 , KS test) or as involved in translation (FDR q value - 1 .26X 10 ' ⁇ GOrilla (Eden et al, 2007)) ( Figure 18C and Table SI).
  • Prpf40a 5.0238077 pos 50 1076 475 1088
  • Tlr4 (rank 10) and its co-receptors Ly96 (MD2) (rank 2) and Cdl4 (rank 3); well-known members of LPS/Tlr4 signaling, including Ticam2 (TRAM, rank 5), Ticaml (TRIP, rank 8), Myd88 (rank 4), Tirap (rank 9), and Traf6 (rank 13); Rela (rank 11), a component of NFKB, which regulates Tnf transcription; and two regulators of NFKB: Ikbkb and Ikbkg (NEMO) (rank 23 and rank 84, respectively).
  • top-ranking genes are highly enriched for known regulators, they also include genes that were not previously associated with either Tlr4 signaling, the LPS response, or regulation of Tnf.
  • Tlr4 signaling the LPS response
  • Tnf the regulatory protein glycosyiation
  • eight bone-fide members of the Tlr4 signaling pathway
  • the other two genes are subunits of the oiigosaccharyltransferase complex (OSTc), a complex responsible for protein glycosyiation (discussed below).
  • OSTc oiigosaccharyltransferase complex
  • Some of these highly ranked genes could be novel regulators, but others may be false positives. [00189] To distinguish these possibilities and empirically determine the true positive rate in the top hits.
  • Tnf levels Applicants gated on live cells, and excluded from the analysis sgRNAs that cause significant reduction in viability (Table 87, Experimental Procedures).
  • PdcdlO CCM3
  • Ppp2 1 a fourth negative regulator
  • Calyculin A a drug that inhibits the protein phosphatase 1 and protein phosphatase 2A complexes, of which Ppp2rla is a member, was previously shown to induce TNF secretion (Boehringer et al., 1999).
  • a secondary pooled screen uncovers additional regulators with greater sensitivity and specificity [00195]
  • the analysis shows that a genome-wide screen can successfully recover known and novel regulators, and suggests that (for the positive regulators), noise in the screen may arise primarily from the large number of sgRNAs screened in a pool in a limited number of cells.
  • Applicants performed a secondary pooled screen targeting 2,569 genes based on the top ranked genes from the genome-wide screen with each of 10 different sgRNAs (with the improved design of (Doench et al., 2014)).
  • the new library contained 28,010 sgRNA (1.66-fold more sgRNAs per gene than the primary screen), including 2,500 non-targeting sgRNAs controls.
  • Applicants used the same number of cells (200X106), resulting in 4.9 -fold more cells per sgRNA..
  • the secondary screen yielded more specific and sensitive results.
  • a greater proportion of the known regulators were even more highly enriched in the Tnf-Low vs. Tnf-high libraries, ( Figure 24A and 24B).
  • Iraki ranked 9 in the secondary screen vs. 187 in the primary screen
  • Irak4 ranked 60 versus 992
  • Sharpin another subunit of the LUBAC complex, ranked 36
  • NEDD8 ranked 52
  • Ube2f ranked 25
  • Neddylation was shown to affect the secretion of cytokines by dendritic cells as well as their ability to activate T cells (Mathewson et al., 2013).
  • the secondary screen overcame some intrinsic limitations of the primary screen, including insufficient numbers of sgRNAs per genes and cells per sgRNAs.
  • the secondary screen follows up a genome -wide screen with a higher depth secondary screen thus represents an effective strategy for maximizing CRISPR-based pooled screens in any biological system that has limits on the number of cells per screen.
  • Positive Tnf regulators are organized in functional modules by their impact on RNA and protein expression [00198] While all the validated regulators affect Tnf levels, the pathways and mechanisms through whic they act may be distinct. To help determine those, Applicants first measured the impact of the validated positive regulators on the expression of four additional protein markers at 8 hours post-LPS (Experimental Procedures), each reflecting distinct facets of BMDCs biology: Cdl lc (the defining surface marker of BMDCs), Cdl 4 (a Tlr4 co-receptor), Mipl a (an induced chemokine), and 116 (an induced inflammatory cytokine).
  • Cdl lc the defining surface marker of BMDCs
  • Cdl 4 a Tlr4 co-receptor
  • Mipl a an induced chemokine
  • 116 an induced inflammatory cytokine
  • Module ⁇ consisted of sgl As targeting 17 genes, including 12 known regulators validated in the screen (Cdl4, Ly96, Cebpb, Ikbkb, Ikbkg, Myd88, Ticaml, Ticam2, TrafZ, TraB), each reducing the levels of Cdl4 and 116, but not Cdl 1 c ( Figure 20A-C and Table SI 4). This is consistent with the known regulators' specific role in activating LPS signaling.
  • Module II included nine genes that each reduced the levels of all four proteins (Figure 20A and Table Si 4).
  • Module III consisted of sgRNAs targeting 4 regulators, each reducing Cdl lc, and 116 expression, but had a very minor, albeit consistent, effect on Mi la (Figure 20B,C) and no effect on CD 14. Among these were members of the PAF complex (below), as well as Polr2g ( Figure 20A-C and Table S14), the only transcriptional and splicing regulator (among 6 Applicants tested here; Table S2) with this phenotype. PAFc and Pol2 are known to physically and functionally interact (Wade et al,, 1996).
  • the modules are further supported by their organization in the context of the known LP8/TLR4 circuit and overall protein interaction network (Figure 29),
  • the PAFc module is supported by known interactions and putatively connected to the core netwOrk through physical interactions with key TPs in the response.
  • individual factors "hit" in the screen are placed in context of known parts of the network, suggesting the basis for their impact.
  • the validated regulators e.g., members of the OSTc
  • Components of the OST complex and the ER folding and translocation pathway are important or Tnf expression in response to LPS sensing
  • oligosaecharyltransferase complex (OSTc) are among the top 100 ranked positive regulators: Dad! , Ddost/OST48, Rpnl, and Rpn2 in ranks 6 7, 14 and 73, respectively, with each validated individually (Figure 20C and Figure 21), and all four are members of the same module by both protein ( Figure 2 ⁇ ) and RNA-Seq ( Figure 20D-F) analysis.
  • OSTc oligosaecharyltransferase complex
  • Each of these 4 hits is an essential subunit for OSTc activity, while the remaining 5 known subunits appear to confer substrate selectivity (Mohorko et al, 2014), with some functional redundancy among 4 of these 5 (Aebi, 2013) (also scoring much lower, ranks 304-7,677 in the screen).
  • the ER-resident OSTc tags asparagine residues of newly-translated proteins with oligosaccharide chains that are critical for protein folding and transport through the ER. Consistent with the identification of OSTc, many other genes that are essential for the ER transport pathway (upstream or downstream of OSTc) were also among the top-ranking validated positive regulators (although not necessarily the OSTc module), including: (1) Srp54c (rank 17, Figure 21 A), a component of the SRP complex that interacts with the hydrophobic N terminus of the protein during protein synthesis and directs it to (2) Srpr (rank 55, Figure 21A), a receptor on the ER membrane (Wild et al., 2004); (3) Sec61 (rank 101 DE rank and 461 ZS; not tested individually), a component of the ER channel through which proteins are translocated (Dudek et al., 2014); (4) Alg2 (rank 87 DE; 269 ZS, Figure 21 A) one of the enzymes necessary for
  • Over 2,300 proteins are known to be N-glycosylated (Zielinska et al., 2010) and knocking out subunits of OSTc may affect Tnf levels directly or indirectly, and - in either case - could reflect a more global effect on N -glycosylated proteins and cell phenotype in LPS- stimulated BMDCs (Notably, there are no discernable differences in viability in the OSTc targeted cells pre- LPS).
  • both LY96 and Tlr4 are -glycosylated, and Tlr4 transport to the membrane is disrupted in the absence of tagged asparagines (da Silva Correia and Ulevitch, 2002),
  • HSP90b above was shown to be important for Tlr4 folding and trafficking, with Tnf levels reduced in response to LPS in HSP90b-null macrophages (Lee et al., 2013; Randow and Seed, 2001 ; Yang et al, 2007).
  • OSTc and additional components of the broader pathway, such as Alg2 could affect Tnf levels by impacting Tlr4 and/or its signaling.
  • Additional regulators with the same profile as OSTc may regulate Tnf through related pathways. These include Hsp90b and Alg2, both of which are known members of the protein folding and secretion pathways (above), and Tmem258 ( Figure 20C and Figure 26).
  • human Tmem258, a molecuiariy uncharacterized gene resides in a locus associated with Crohn's disease in Genome Wide Association Studies (Franke et a!., 2010) and was reported to be targeted by ANRIL, a long non coding RNA associated with immune and metabolic diseases (Bochenek et al., 2013).
  • Tmem258 induces the same ER stress genes pre-LPS as does targeting of OSTc (5/14 genes; 7.7X10 "3 Q-FDR GOrrila; Table S14), and has a similar impact on the protein markers and RNA-Seq profiles post-LPS, suggesting that a similar mechanism may underlie its function.
  • the PAF complex and its physical interactors form a module that positively regulates Tnf protein expression
  • Cdc73 (rank 842 in the primary screen) was likely a false negative, since two additionally-designed sgR As did reduce Tnf expression (Figure 27B). Furthermore, the Ash21 subunit of the MLL complex, previously reported to physically interact with Cdc73 (Rozenblatt-Rosen et al., 2005), was also validated as a positive regulator of Tnf in the screen (rank 41, Figure 27B). Both PAFc and MLL can affect H3 9 methylation (Krogan et al., 2003; Steward et al., 2006; Taniue et al., 201 1 ).
  • PAFc is known to interact with RNA polymerase II with roles in transcription elongation and 3 ' mRNA processing (Jaehning, 2010), and regulation of transcription elongation was previously shown to be an important key step in the DC transcriptional response (Beaudoin and jaffrin, 1989; Hargreaves et ai, 2009).
  • Prior studies have implicated Paf-1 or PAFc in regulation of antiviral gene expression either as an activator (Marazzi et al., 2012) following viral infection, or as a suppressor (Fonseca et al, 2013), with (Fonseca et al., 2013) or without (Kim et al., 2013a) viral infection.
  • PAFc was not previously implicated in Tnf or inflammatory gene expression
  • LPS is known to induce the expression of both anti-viral and pro inflammatory genes (Am.it et al, 2009),
  • AUH binds AU-rich motifs in 3'UTRs, and the stability of the Tnf transcript is known to be regulated through such an AU rich motif by three other RNA binding proteins: AUF1 ( habar, 2010), Zfp36 (Carballo et aL 1998) and HuR (Dean et a!., 2001 ; Tiedje et a!., 2012). It would be interesting to explore if AUH also interacts with the 3 'UTR of Tnf directly to regulate Tnf levels.
  • Example 7 Ex vivo genome editing in primary cells by lentiviral-mediated sgRNA expression
  • the CRISPR-CAS9 screen in primary cells described herein can be used in different primary ceil types, including, but not limited to macrophages and T cells.
  • the primary ceils may be immune cells, endocrine cells, nervous system cells, muscle cells, exocrine secretory epithelial ceils, hormone secreting cells, or metabolism and storage cells.
  • Primary cells refer to any cells that are cultured directly from a subject. Different conditions can be used, such as, activation with another pathogenic signal or in the presence of cytokines.
  • Applicants use other markers in addition to TNF as a phenotypic readout, including, but not limited to transcription factors, cell surface molecules, secreted cytokines, and secreted hormones.
  • immunoprecipitation approach reveals principles of dynamic gene regulation in mammals.
  • ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109, E2579- 2586.
  • LKB1 modulates lung cancer differentiation and metastasis. Nature 448, 807-810.

Abstract

L'invention concerne un procédé permettant de moduler l'activité des leucocytes, consistant à administrer à un leucocyte un vecteur contenant une ou des molécules d'acides nucléiques, grâce à quoi le leucocyte renferme Cas9 et le vecteur exprime un ou plusieurs ARN afin de guider le Cas9 vers l'introduction de mutations dans un ou plusieurs loci génétiques cibles dans le leucocyte, ce qui permet de moduler l'expression d'un ou plusieurs gènes exprimés dans le leucocyte. L'invention consiste également à identifier des gènes associés à des réponses des leucocytes et à modéliser expérimentalement l'activation aberrante des leucocytes et des maladies associées aux leucocytes par l'introduction de mutations dans les leucocytes. L'invention comprend l'analyse de traitements putatifs avec ces modèles, par exemple, l'analyse de composés chimiques putatifs qui peuvent être pertinents du point de vue pharmaceutique pour le traitement ou de thérapie génique qui peut être pertinente pour un traitement, ou leurs combinaisons. L'invention permet l'étude de maladies génétiques et de traitements putatifs pour mieux comprendre et soulager des maladies associées aux leucocytes.
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